Fed-batch methods for producing adalimumab

ABSTRACT

The invention describes improved methods and compositions for producing a recombinant protein, e.g., an antibody, in mammalian cell culture. In addition, the invention provides improved cell culture media, including improved production media, feed solutions, and combination feeds, which may be used to improve protein productivity in mammalian cell culture.

RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.13/308,075, filed Nov. 30, 2011, now U.S. Pat. No. 8,663,995, which is aContinuation of U.S. patent application Ser. No. 11/901,274, filed Sep.13, 2007, now U.S. Pat. No. 8,093,045, issued Jan. 10, 2012, whichclaims the benefit of priority to U.S. Provisional Application No.60/845,158, filed on Sep. 13, 2006, and U.S. Provisional Application No.60/876,374, filed on Dec. 21, 2006. The contents of each of the abovepriority documents is incorporated by reference herein. This applicationalso incorporates by reference U.S. Pat. Nos. 8,093,045 and 8,663,995and U.S. Patent Application Publication Nos. 2008/0227136 and2012/0077213 in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 11, 2013, isnamed Sequence Listing and is 4 kilobytes in size.

BACKGROUND OF THE INVENTION

Recombinant DNA technology has provided a means to produce proteins inamounts which allow for their use in a spectrum of applications,including therapeutic, diagnostic, agricultural, and research purposes.

One goal of recombinant protein production is the optimization of cellculture media and conditions in order to obtain the greatest amount ofprotein and the most efficient means of productivity. Any improvement,including incremental improvements, can have enormous benefitseconomically. In the pharmaceutical industry, optimization of proteinproduction for biologics used in therapies for the treatment of diseaseis advantageous, as any improvement can have significant impact when thebiologic is manufactured on a large scale. As such, there remains a needto maximize protein production from cell cultures expressing biologicproteins for use in medicine.

Typically mammalian cell culture media is based on commerciallyavailable media formulations, including, for example, DMEM or Ham's F12.Often media formulations are not sufficiently enriched to supportincreases in both cell growth and biologic protein expression. Thereremains a need for improved cell culture media, supplements, and cellculture methods for improved protein production.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for improving proteinexpression in cell culture, particularly mammalian cell culture. Theinvention relates to improved cell culture media, including media forgrowing cells for protein expression and cell culture production mediaoptimized for protein expression.

The invention also includes optimized methods and media formulations forhigh protein expression in mammalian cell culture. In particular, thecell culture media is optimized for expression of antibodies inmammalian cell culture, e.g., CHO cells. Improved fed batch methods andcompositions for promoting protein production by adding supplementalsolutions, e.g., hydrolysate containing solutions and concentrationbasal media solutions, are also provided.

The invention provides improved salt-free basal growth media for use inmammalian cell culture. The invention includes a serum-free cell culturemedium comprising Part A, Part B, and Part C, wherein Part A consistsessentially of a modified basal medium which excludes the followingcomponents: sodium bicarbonate, a buffer, mono-basic sodium phosphate,di-basic sodium phosphate, an osmolarity regulator, a surfactant, andmonosaccharide glucose; Part B consists essentially of an inorganic ironsource; and Part C comprises a recombinant growth factor; a buffer; anosmolarity regulator; an energy source; and at least two differentnon-animal hydrolysates.

In one embodiment, Part A further comprises non-ferrous metal ions,vitamins, or a combination of both. In one embodiment, the inorganiciron source of Part B is ferric citrate e.g., about 100-150 mg/L or0.1-1 mM final solution concentration ferric citrate. In anotherembodiment, the inorganic iron source of Part B is ferric citrate e.g.,about 122.5 mg/L or 0.5 mM final solution concentration ferric citrate.

In one embodiment, the recombinant growth factor of Part C is selectedfrom the group consisting of insulin or a recombinant analog, IGF-1, anda combination of insulin and IGF-1, e.g., about 4 mg/L to 13 mg/Linsulin, or a recombinant analog thereof.

In one embodiment, the buffer which is excluded from the modified basalmedium is HEPES buffer.

In one embodiment, the buffer of Part C comprises a phosphate buffer,HEPES, and sodium bicarbonate, e.g., about 0.1 to 3 g/L sodiumbicarbonate, about 0.1 to 3 g/L HEPES. In one embodiment, the buffer ofPart C comprises 1.6 g/L of sodium bicarbonate and/or about 1.8 g/LHEPES. In one embodiment, the phosphate buffer comprises about 0.01 to0.5 g/L mono- and di-basic sodium phosphates.

In a further embodiment, Part C further comprises asparagine, glutamine,or glutamine and asparagine.

In one embodiment, the osmolarity regulator of Part C is NaCl, e.g.,about 1.0 to 6.5 g/L NaCl.

In one embodiment, the energy source of Part C is a monosaccharide,e.g., glucose (such as D-glucose), maltose, mannose, galactose andfructose. In one embodiment, the cell culture medium of the inventioncomprises no greater than about 7.0 g/L glucose.

In another embodiment, the cell culture medium of the inventioncomprises at least two different non-animal based hydrolysates of Part Care a plant-based hydrolysate and a hydrolysate which is neither animalor plant-based. An example of a plant-based hydrolysate that may be usedin the invention is a soy-based hydrolysate. An example of a hydrolysatethat is neither animal or plant-based is a yeast-based hydrolysate.

In one embodiment, the cell culture medium of the invention furthercomprises methotrexate. In one embodiment, the cell culture mediumfurther comprises about 100 nM to 5000 nM methotrexate.

In yet another embodiment, the cell culture medium further comprises acell protectant or surfactant. An example of a surfactant that may beused in the cell culture medium of the invention is methyl cellulose ora pluronic polyol, e.g., Pluronic F-68. In one embodiment, the cellculture medium comprises about 0.1-5 g/L Pluronic F-68. In oneembodiment, the cell culture medium comprises about 1.0 g/L PluronicF-68.

In still another embodiment of the invention, the cell culture mediumfurther comprises L-glutamine.

In one embodiment, the cell culture medium has a pH range from 7.1 to7.3.

In another embodiment, the cell culture medium of the invention has anosmolality ranging from about 320 to 450 mOsm/kg.

The invention includes a serum-free cell culture medium comprising: abasal medium; about 8-12 ml/kg or 116-126 mg/L ferric citrate; about 2-6mg/kg recombinant human insulin; about 2-5 g/kg anhydrous glucose; about0.1-0.5 g/kg L-glutamine; about 1-3 g/kg sodium bicarbonate; about0.01-0.05 g/kg NaH₂PO₄.H₂O; about 0.4 to 0.5 g/kg of Na₂HPO₄.7H₂O; andabout 1.0-3.0 g/kg yeast-based hydrolysate. In one embodiment, the cellculture medium comprises a basal medium; about 10.0 ml/kg or 122 mg/Lferric citrate; about 4.0 mg/kg recombinant human insulin; about 3.5g/kg anhydrous glucose; about 0.29 g/kg L-glutamine; about 1.6 g/kgsodium bicarbonate; about 0.03 g/kg NaH₂PO₄.H₂O; about 0.43 to 0.44 g/kgof Na₂HPO₄.7H₂O; and about 2.0 g/kg yeast-based hydrolysate. In oneembodiment, the cell culture medium consists essentially of a basalmedium; about 10.0 ml/kg or 122 mg/L ferric citrate; about 4.0 mg/kgrecombinant human insulin; about 3.5 g/kg anhydrous glucose; about 0.29g/kg L-glutamine; about 1.6 g/kg sodium bicarbonate; about 0.03 g/kgNaH₂PO₄.H₂O; about 0.43 to 0.44 g/kg of Na₂HPO₄.7H₂O; and about 2.0 g/kgyeast-based hydrolysate.

The invention further provides a serum-free cell culture mediumconsisting essentially of a basal medium; about 8-12 ml/kg or 116-126mg/L ferric citrate; about 2-6 mg/kg recombinant human insulin; about2-5 g/kg anhydrous glucose; about 0.1-0.5 g/kg L-glutamine; about 1-3g/kg sodium bicarbonate; about 0.01-0.05 g/kg NaH₂PO₄.H₂O; about 0.4 to0.5 g/kg of Na₂HPO₄.7H₂O; and about 1.0-3.0 g/kg yeast-basedhydrolysate. In one embodiment, the cell culture consists essentially ofa basal medium; about 8-12 ml/kg or 116-126 mg/L ferric citrate; about2-6 mg/kg recombinant human insulin; about 2-5 g/kg anhydrous glucose;about 0.1-0.5 g/kg L-glutamine; about 1-3 g/kg sodium bicarbonate; about0.01-0.05 g/kg NaH₂PO₄.H₂O; about 0.4 to 0.5 g/kg of Na₂HPO₄.7H₂O; andabout 1.0-3.0 g/kg yeast-based hydrolysate

In one embodiment, the cell culture medium further comprises about 2.50mL/kg methotrexate.

The invention also includes a method for producing a protein comprisingculturing mammalian cells comprising a nucleic acid encoding the proteinin the culture medium of the invention; and transferring the culture ofinto a cell culture production medium, such that the protein isproduced.

In one embodiment, the protein is an antibody, including for example,D2E7 (adalimumab).

The invention further provides a serum-free cell culture productionmedium comprising: a modified basal medium which excludes the followingcomponents sodium bicarbonate, buffer, mono-basic sodium phosphate,di-basic sodium phosphate, an osmolarity regulator, a surfactant, andmonosaccharide glucose; about 8 to 12 ml/kg or 122.45 mg/L ferriccitrate; about 4 to 8 mL/kg or 10 to 14 mg/kg recombinant human insulin;about 5 to 9 g/kg anhydrous glucose; about 0.5 to 0.7 g/kg L-glutamine;about 1 to 2 g/kg sodium bicarbonate; about 1 to 2 g/kg HEPES; about 2to 3 g/kg NaCl; about 0.5 to 2 g/kg Pluronic F-68; about 0.01 to 0.1g/kg NaH₂PO₄.H₂O; about 0.4 to 0.5 g/kg Na₂HPO₄.7H₂O; about 8 to 12 g/kgyeast-based hydrolysate; and about 60 to 70 g/kg plant-basedhydrolysate. In one embodiment, the cell culture production mediumconsists essentially of a modified basal medium which excludes thefollowing components sodium bicarbonate, buffer, mono-basic sodiumphosphate, di-basic sodium phosphate, an osmolarity regulator, asurfactant, and monosaccharide glucose; about 8 to 12 ml/kg or 122.45mg/L ferric citrate; about 4 to 8 mL/kg or 10 to 14 mg/kg recombinanthuman insulin; about 5 to 9 g/kg anhydrous glucose; about 0.5 to 0.7g/kg L-glutamine; about 1 to 2 g/kg sodium bicarbonate; about 1 to 2g/kg HEPES; about 2 to 3 g/kg NaCl; about 0.5 to 2 g/kg Pluronic F-68;about 0.01 to 0.1 g/kg NaH₂PO₄.H₂O; about 0.4 to 0.5 g/kg Na₂HPO₄.7H₂O;about 8 to 12 g/kg yeast-based hydrolysate; and about 60 to 70 g/kgplant-based hydrolysate. In another embodiment, the cell cultureproduction medium comprises a basal medium, about 10.0 ml/kg or 122.45mg/L ferric citrate; about 6.0 mL/kg or 12 mg/kg recombinant humaninsulin; about 7.0 g/kg anhydrous glucose; about 0.58 to 0.59 g/kgL-glutamine; about 1.6 g/kg sodium bicarbonate; about 1.8 g/kg HEPES;about 2.4 to 2.5 g/kg NaCl; about 1.0 g/kg Pluronic F-68; about 0.03 to0.04 g/kg NaH₂PO₄.H₂O; about 0.43 to 0.44 g/kg Na₂HPO₄.7H₂O; about 10.7g/kg yeast-based hydrolysate; and about 6.9 to 7.0 g/kg plant-basedhydrolysate.

The invention also provides a serum-free cell culture medium comprisinga modified basal medium, which excludes the following components sodiumbicarbonate, buffer, mono-basic sodium phosphate, di-basic sodiumphosphate, an osmolarity regulator, a surfactant, and monosaccharideglucose; about 8 to 12 ml/kg or 122.45 mg/L ferric citrate; about 3 to 5mL/kg or 6 to 8 mg/kg recombinant human insulin; about 5 to 9 g/kganhydrous glucose; about 0.1 to 2 g/kg L-glutamine; about 1 to 2 g/kgsodium bicarbonate; about 1 to 2 g/kg HEPES; about 2 to 3 g/kg NaCl;about 0.1 to 2 g/kg Pluronic F-68; about 0.01 to 0.1 g/kg NaH₂PO₄.H₂O;about 0.4 to 0.5 g/kg Na₂HPO₄.7H₂O; about 0.4 to 0.5 g/kg L-asparaginemonohydrate; about 2 to 6 g/kg yeast-based hydrolysate; and about 2 to 4g/kg plant-based hydrolysate. In one embodiment, the cell culture mediumconsists essentially of a modified basal medium, which excludes thefollowing components sodium bicarbonate, buffer, mono-basic sodiumphosphate, di-basic sodium phosphate, an osmolarity regulator, asurfactant, and monosaccharide glucose; about 8 to 12 ml/kg or 122.45mg/L ferric citrate; about 3 to 5 mL/kg or 6 to 8 mg/kg recombinanthuman insulin; about 5 to 9 g/kg anhydrous glucose; about 0.1 to 2 g/kgL-glutamine; about 1 to 2 g/kg sodium bicarbonate; about 1 to 2 g/kgHEPES; about 2 to 3 g/kg NaCl; about 0.1 to 2 g/kg Pluronic F-68; about0.01 to 0.1 g/kg NaH₂PO₄.H₂O; about 0.4 to 0.5 g/kg Na₂HPO₄.7H₂O; about0.4 to 0.5 g/kg L-asparagine monohydrate; about 2 to 6 g/kg yeast-basedhydrolysate; and about 2 to 4 g/kg plant-based hydrolysate. In oneembodiment, the cell culture medium comprises a modified basal medium;about 10.0 ml/kg or 122.45 mg/kg ferric citrate; about 3.8 to 3.9 mL/kgor 7.8 mg/kg recombinant human insulin; about 7.0 g/kg anhydrousglucose; about 0.8 to 0.9 g/kg L-glutamine; about 1.6 g/kg sodiumbicarbonate; about 1.8 g/kg HEPES; about 2.6 to 2.7 g/kg NaCl; about 1.0g/kg Pluronic F-68; about 0.03 to 0.04 g/kg NaH₂PO₄.H₂O; about 0.43 to0.44 g/kg Na₂HPO₄.7H₂O; about 0.45 g/kg L-asparagine monohydrate; about4.0 g/kg yeast-based hydrolysate; and about 2.6 g/kg plant-basedhydrolysate.

The invention also includes a serum-free cell culture medium comprisinga modified basal medium, which excludes the following components sodiumbicarbonate, buffer, mono-basic sodium phosphate, di-basic sodiumphosphate, an osmolarity regulator, a surfactant, and monosaccharideglucose; about 8 to 10 ml/kg or 120 to 130 mg/L ferric citrate; about 3to 5 mL/kg or 7.8 mg/kg recombinant human insulin; about 5 to 9 g/kganhydrous glucose; about 0.8 to 0.9 g/kg L-glutamine; about 0.3 to 0.5g/kg L-asparagine monohydrate; about 1 of 2 g/kg sodium bicarbonate;about 1 to 2 g/kg HEPES; about 2 to 3 g/kg NaCl; about 0.5 to 2 g/kgPluronic F-68; about 0.01 to 0.1 g/kg NaH₂PO₄.H₂O; about 0.1 to 1.0 g/kgNa₂HPO₄.7H₂O; about 2 to 6 g/kg yeast-based hydrolysate; and about 2 to4 g/kg plant-based hydrolysate. In one embodiment, the cell culturemedium consists essentially of a modified basal medium, which excludesthe following components sodium bicarbonate, buffer, mono-basic sodiumphosphate, di-basic sodium phosphate, an osmolarity regulator, asurfactant, and monosaccharide glucose; about 8 to 10 ml/kg or 120 to130 mg/L ferric citrate; about 3 to 5 mL/kg or 7.8 mg/kg recombinanthuman insulin; about 5 to 9 g/kg anhydrous glucose; about 0.8 to 0.9g/kg L-glutamine; about 0.3 to 0.5 g/kg L-asparagine monohydrate; about1 of 2 g/kg sodium bicarbonate; about 1 to 2 g/kg HEPES; about 2 to 3g/kg NaCl; about 0.5 to 2 g/kg Pluronic F-68; about 0.01 to 0.1 g/kgNaH₂PO₄.H₂O; about 0.1 to 1.0 g/kg Na₂HPO₄.7H₂O; about 2 to 6 g/kgyeast-based hydrolysate; and about 2 to 4 g/kg plant-based hydrolysate.In another embodiment, the cell culture medium comprises a modifiedbasal medium; about 10 ml/kg or 122 mg/L ferric citrate; about 3.8 to3.9 mL/kg or 7.8 mg/kg recombinant human insulin; about 7.0 g/kganhydrous glucose; about 0.87 to 0.88 g/kg L-glutamine; about 0.45 g/kgL-asparagine monohydrate; about 1.6 g/kg sodium bicarbonate about 1.8g/kg HEPES; about 2.67 to 2.68 g/kg NaCl; about 1.0 g/kg Pluronic F-68;about 0.03 to 0.04 g/kg NaH₂PO₄.H₂O; about 0.43 to 0.44 g/kgNa₂HPO₄4.7H₂O; about 4.0 g/kg yeast-based hydrolysate; and about 2.6g/kg plant-based hydrolysate.

The invention includes a serum-free cell culture medium comprising basalcell growth medium; about 8 to 12 ml/kg or 120 to 130 mg/L ferriccitrate; about 2 to 6 mg/kg recombinant human insulin; about 150 to 250g/kg anhydrous glucose; about 0.1 to 0.5 g/kg L-glutamine; about 1 to 2g/kg sodium bicarbonate; and about 5 to 15 g/kg yeast-based hydrolysate.In one embodiment, the cell culture medium consists essentially of basalcell growth medium; about 8 to 12 ml/kg or 120 to 130 mg/L ferriccitrate; about 2 to 6 mg/kg recombinant human insulin; about 150 to 250g/kg anhydrous glucose; about 0.1 to 0.5 g/kg L-glutamine; about 1 to 2g/kg sodium bicarbonate; and about 5 to 15 g/kg yeast-based hydrolysate.In a further embodiment, the cell culture medium comprises basal cellgrowth medium; about 10 ml/kg or 122.45 mg/L ferric citrate; about 4mg/kg recombinant human insulin; about 200 g/kg anhydrous glucose; about0.29 to 0.30 g/kg L-glutamine; about 1.6 g/kg sodium bicarbonate; andabout 11 g/kg yeast-based hydrolysate. In an additional embodiment, theprotein is an antibody, including, for example a fully human, anti-IL-12antibody, e.g., ABT-874.

The invention also includes a serum-free cell culture medium comprisingbasal cell growth medium; about 8 to 12 ml/kg or 120 to 130 mg/L ferriccitrate; about 2 to 6 mg/kg recombinant human insulin; about 1 to 3 g/kganhydrous glucose; about 0.1 to 1 g/kg L-glutamine; about 1 to 2 g/kgsodium bicarbonate; and about 1 to 4 g/kg yeast-based hydrolysate. Inone embodiment, the cell culture medium consists essentially of basalcell growth medium; about 8 to 12 ml/kg or 120 to 130 mg/L ferriccitrate; about 2 to 6 mg/kg recombinant human insulin; about 1 to 3 g/kganhydrous glucose; about 0.1 to 1 g/kg L-glutamine; about 1 to 2 g/kgsodium bicarbonate; and about 1 to 4 g/kg yeast-based hydrolysate. Inanother embodiment, the cell culture medium comprises a basal cellgrowth medium; about 10 ml/kg or 122.45 mg/L ferric citrate; about 4mg/kg recombinant human insulin; about 1.5 g/kg anhydrous glucose; about0.29 to 0.30 g/kg L-glutamine; about 1.6 g/kg sodium bicarbonate; andabout 2 g/kg yeast-based hydrolysate. In one embodiment, the pH of thecell culture medium is about 7.10 to 7.30 and the osmolality ranges fromabout 300 to 340 mOsm/kg. In still another embodiment, the cell culturemedium comprises at least 8 g/kg yeast-based hydrolysate. In oneembodiment, the protein which is produced in a mammalian cell, e.g., CHOcell, using the cell culture medium is an antibody, including, forexample, an anti-IL-12 antibody or an anti-EPO-R antibody, e.g.,ABT-874.

The invention further provides a cell culture medium comprising amodified basal medium which excludes the following components sodiumbicarbonate, buffer, mono-basic sodium phosphate, di-basic sodiumphosphate, an osmolarity regulator, a surfactant, and monosaccharideglucose; about 8 to 12 ml/kg or 120 to 130 mg/L ferric citrate; about2.5 to 4.5 mL/kg or 7.8 mg/kg recombinant human insulin; about 5 to 9g/kg anhydrous glucose; about 0.5 to 1 g/kg L-glutamine; about 0.1 to 1g/kg L-asparagine monohydrate; about 1 to 2 g/kg sodium bicarbonate;about 1 to 2 g/kg HEPES; about 1 to 4 g/kg NaCl; about 0.1 to 2 g/kgPluronic F-68; about 0.01 to 0.1 g/kg NaH₂PO₄.H₂O; about 0.1 to 1 g/kgNa₂HPO₄.7H₂O; about 2 to 6 g/kg yeast-based hydrolysate; and about 2 to6 g/kg plant-based hydrolysate. In one embodiment, the cell culturemedium of the invention consists essentially of a modified basal mediumwhich excludes the following components sodium bicarbonate, buffer,mono-basic sodium phosphate, di-basic sodium phosphate, an osmolarityregulator, a surfactant, and monosaccharide glucose; about 8 to 12 ml/kgor 120 to 130 mg/L ferric citrate; about 2.5 to 4.5 mL/kg or 7.8 mg/kgrecombinant human insulin; about 5 to 9 g/kg anhydrous glucose; about0.5 to 1 g/kg L-glutamine; about 0.1 to 1 g/kg L-asparagine monohydrate;about 1 to 2 g/kg sodium bicarbonate; about 1 to 2 g/kg HEPES; about 1to 4 g/kg NaCl; about 0.1 to 2 g/kg Pluronic F-68; about 0.01 to 0.1g/kg NaH₂PO₄.H₂O; about 0.1 to 1 g/kg Na₂HPO₄.7H₂O; about 2 to 6 g/kgyeast-based hydrolysate; and about 2 to 6 g/kg plant-based hydrolysate.In another embodiment, the cell culture medium comprises a modifiedbasal medium; about 10 ml/kg or 122.45 mg/L ferric citrate; about 3.8 to3.9 mL/kg or 7.8 mg/kg recombinant human insulin; about 7.0 g/kganhydrous glucose; about 0.87 to 0.88 g/kg L-glutamine; about 0.45 g/kgL-asparagine monohydrate; about 1.6 g/kg sodium bicarbonate; about 1.8g/kg HEPES; about 2.67 g/kg NaCl; about 1.0 g/kg Pluronic F-68; about0.03 to 0.04 g/kg NaH₂PO₄.H₂O; about 0.43 to 0.44 g/kg Na₂HPO₄.7H₂O;about 4.0 g/kg yeast-based hydrolysate; and about 2.6 g/kg plant-basedhydrolysate.

The invention also includes a cell culture production medium comprisinga modified basal medium, which is modified to remove the followingcomponents sodium bicarbonate, HEPES buffer, mono-basic sodiumphosphate, di-basic sodium phosphate, an osmolarity regulator, asurfactant, and monosaccharide glucose; about 8 to 12 ml/kg or 120 to130 mg/L ferric citrate; about 4 to 8 mL/kg or 10 to 14 mg/kgrecombinant human insulin; about 5 to 9 g/kg anhydrous glucose; about0.1 to 1 g/kg L-glutamine; about 1 to 2 g/kg sodium bicarbonate; about 1to 2 g/kg HEPES; about 1 to 3 g/kg NaCl; about 0.5 to 2 g/kg PluronicF-68; about 0.01 to 0.1 g/kg NaH₂PO₄.H₂O; about 0.1 to 1 g/kgNa₂HPO₄.7H₂O; about 8 to 12 g/kg yeast-based hydrolysate; and about 6 to8 g/kg plant-based hydrolysate. In one embodiment, the cell cultureproduction medium of the invention consists essentially of a modifiedbasal medium, which is modified to remove the following componentssodium bicarbonate, HEPES buffer, mono-basic sodium phosphate, di-basicsodium phosphate, an osmolarity regulator, a surfactant, andmonosaccharide glucose; about 8 to 12 ml/kg or 120 to 130 mg/L ferriccitrate; about 4 to 8 mL/kg or 10 to 14 mg/kg recombinant human insulin;about 5 to 9 g/kg anhydrous glucose; about 0.1 to 1 g/kg L-glutamine;about 1 to 2 g/kg sodium bicarbonate; about 1 to 2 g/kg HEPES; about 1to 3 g/kg NaCl; about 0.5 to 2 g/kg Pluronic F-68; about 0.01 to 0.1g/kg NaH₂PO₄.H₂O; about 0.1 to 1 g/kg Na₂HPO₄.7H₂O; about 8 to 12 g/kgyeast-based hydrolysate; and about 6 to 8 g/kg plant-based hydrolysate.In another embodiment, the cell culture production medium comprises amodified basal medium;

about 10 ml/kg or 122.45 mg/L ferric citrate; about 6.0 mL/kg or 12mg/kg recombinant human insulin; about 7.0 g/kg anhydrous glucose; about0.58 to 0.59 g/kg L-glutamine; about 1.6 g/kg sodium bicarbonate about1.8 g/kg HEPES; about 2.45 g/kg NaCl; about 1.0 g/kg Pluronic F-68;about 0.03 to 0.04 g/kg NaH₂PO₄.H₂O; about 0.43 to 0.44 g/kgNa₂HPO₄.7H₂O; about 10.7 g/kg yeast-based hydrolysate; and about 6.9 to7.0 g/kg plant-based hydrolysate.

Another aspect of the invention is a cell culture production mediumcomprising a modified basal medium which excludes the followingcomponents sodium bicarbonate, buffer, mono-basic sodium phosphate,di-basic sodium phosphate, an osmolarity regulator, a surfactant, andmonosaccharide glucose; about 8 to 12 ml/kg or 110 to 130 mg/L ferriccitrate; about 4 to 8 mL/kg or 11 to 15 mg/kg recombinant human insulin;about 5 to 9 g/kg anhydrous glucose; about 0.1 to 1 g/kg L-glutamine;about 1 to 2 g/kg sodium bicarbonate about 1 to 2 g/kg HEPES; about 1 to3 g/kg NaCl; about 0.1 to 2 g/kg Pluronic F-68; about 0.01 to 0.1 g/kgNaH₂PO₄.H₂O; about 0.1 to 1 g/kg Na₂HPO₄.7H₂O; about 12 to 16 g/kgyeast-based hydrolysate; and about 8 to 10 g/kg plant-based hydrolysate.In one embodiment, the cell culture production medium consistsessentially of a modified basal medium which excludes the followingcomponents sodium bicarbonate, buffer, mono-basic sodium phosphate,di-basic sodium phosphate, an osmolarity regulator, a surfactant, andmonosaccharide glucose; about 8 to 12 ml/kg or 110 to 130 mg/L ferriccitrate; about 4 to 8 mL/kg or 11 to 15 mg/kg recombinant human insulin;about 5 to 9 g/kg anhydrous glucose; about 0.1 to 1 g/kg L-glutamine;about 1 to 2 g/kg sodium bicarbonate about 1 to 2 g/kg HEPES; about 1 to3 g/kg NaCl; about 0.1 to 2 g/kg Pluronic F-68; about 0.01 to 0.1 g/kgNaH₂PO₄.H₂O; about 0.1 to 1 g/kg Na₂HPO₄.7H₂O; about 12 to 16 g/kgyeast-based hydrolysate; and about 8 to 10 g/kg plant-based hydrolysate.In another embodiment, the cell culture production medium of theinvention comprises a modified basal medium; about 10 ml/kg or 122.45mg/L ferric citrate; about 6.5 mL/kg or 13 mg/kg recombinant humaninsulin; about 7.0 g/kg anhydrous glucose; about 0.58 to 0.59 g/kgL-glutamine; about 1.6 g/kg sodium bicarbonate; about 1.8 g/kg HEPES;about 2.45 g/kg NaCl; about 1.0 g/kg Pluronic F-68; about 0.03 to 0.04g/kg NaH₂PO₄.H₂O; about 0.43 to 0.44 g/kg Na₂HPO₄.7H₂O; about 14.2 to14.3 g/kg yeast-based hydrolysate; and about 9.2 to 9.3 g/kg plant-basedhydrolysate.

In one embodiment, the cell culture medium has a pH of about 6 to 8. Inanother embodiment, the cell culture medium has a pH of about 7.10 to7.20.

In one embodiment, the cell culture medium has osmolality of about 350to 450 mOsm/kg. In another one embodiment, the cell culture medium hasosmolality of about 373 to 403 mOsm/kg.

The cell culture media of the invention may further comprisemethotrexate. In one embodiment, the cell culture medium furthercomprises methotrexate, e.g., about 1-10 mL/kg. In another embodiment,the cell culture medium further comprises methotrexate, e.g., about 2.50mL/kg

In one embodiment, the protein which is expressed in the cell culture isan antibody, or antigen-binding fragment thereof. In one embodiment, theantibody, or antigen-binding fragment thereof, is an anti-TNFα antibodyor an anti-EPO-R antibody. In another embodiment, the anti-TNFαantibody, or antigen-binding fragment thereof, is a fully humananti-TNFα antibody, including, for example, the fully human anti-TNFαantibody is D2E7 (adalimumab). In yet another embodiment, the antibody,or antigen-binding fragment thereof, is an anti-IL-12 or an anti-IL-18antibody, including a fully human anti-IL-12 or an anti-IL-18 antibody.

The invention also includes a method of producing a protein, e.g., anantibody or antigen-binding portion thereof, comprising culturing amammalian cell comprising a nucleic acid encoding the protein, e.g.,antibody, in a cell culture medium presented herein. In one embodiment,the cell culture medium is a cell culture production medium. Examples ofantibodies, or antigen binding fragments thereof, which may be producedusing the methods and compositions of the invention include ananti-IL-18 antibody, an anti-TNFα antibody, an anti-IL-12 antibody, andan anti-EPO receptor (EPO-R) antibody.

In one embodiment, the invention further comprises isolating the proteinfrom the cell culture media, e.g., cell culture production media,described herein.

In one embodiment, the cell culture media and methods of the inventionare for culturing mammalian cells, including Chinese Hamster Ovary (CHO)cells.

The invention also includes a Chinese Hamster Ovary (CHO) cell in any ofthe cell culture media described herein.

The invention also provides an improved fed batch method and relatedcell culture media for producing proteins in mammalian cell culture,e.g., CHO cells. One aspect of the invention is a fed batch method ofproducing a protein comprising culturing mammalian cells comprising anucleic acid encoding the protein in a cell culture comprising a cellculture production medium; and feeding the mammalian cells by adding ahydrolysate enrichment solution and a basal enrichment solution to thecell culture during a time period, wherein the hydrolysate enrichmentsolution comprises at least two different non-animal-based hydrolysates,such that the protein is produced.

In one embodiment, the basal enrichment solution comprises aconcentrated basal medium. In another embodiment, the basal enrichmentsolution comprises a basal medium, asparagine, and glucose. In stillanother embodiment, the basal medium is PF CHO.

In one embodiment, the hydrolysate enrichment solution comprises a firsthydrolysate which is not derived from a plant or an animal and a secondplant-based hydrolysate. In one embodiment, the hydrolysate which is notderived from a plant or an animal and a plant-based hydrolysate is ayeast-based hydrolysate. In one embodiment, the plant-based hydrolysateis a soy-based hydrolysate.

In one embodiment, the protein which is produced is an antibody, orantigen binding portion thereof. Examples of antibodies, orantigen-binding portions thereof, which may be used in the fed batchmethods of the invention include an anti-TNFα antibody, an anti-IL-12antibody, an anti-IL-18 antibody, and an anti-EPO receptor (EPO-R)antibody.

The invention includes a fed batch method of producing an anti-TNFαantibody, including, for example a fully human anti-TNFα antibody suchas adalimumab, comprising culturing Chinese Hamster Ovary (CHO) cellscomprising a nucleic acid encoding the anti-TNFα antibody in a cellculture comprising a cell culture production medium; and feeding the CHOcells by adding a hydrolysate enrichment solution and a basal enrichmentsolution to the cell culture during a time period, wherein the basalenrichment solution comprises a basal medium, asparagine, and glucose,and wherein the hydrolysate enrichment solution comprises at least twodifferent non-animal-based hydrolysates, such that the anti-TNFαantibody is produced.

The invention also features a fed batch method of producing an anti-TNFαantibody comprising culturing CHO cells comprising a nucleic acidencoding the anti-TNFα antibody in a cell culture comprising a cellculture production medium comprising a at least 1-5 g/L, e.g., 2.0 g/Lof glucose, wherein the concentration of glucose is controlled by addingglucose to the cell culture production medium as required to maintain aconcentration of at least 1-5 g/L, e.g., 2.0 g/L of glucose; and feedingthe CHO cells by adding a hydrolysate enrichment solution and a basalenrichment solution to the cell culture during a time period, whereinthe basal enrichment solution comprises a basal medium, asparagine, andglucose, and wherein the hydrolysate enrichment solution comprises atleast two different non-animal-based hydrolysates, such that theanti-TNFα antibody is produced.

In one embodiment, the invention includes further recovering theanti-TNFα antibody.

In yet another embodiment, the cell culture is cultured at a temperatureranging from about 32 to 38° C., e.g., 35° C.

In one embodiment, the cell culture production medium is maintainedbetween 20 and 65% dissolved oxygen, e.g., at about 30% dissolvedoxygen.

In one embodiment, the osmolarity of the cell culture production mediumis maintained throughout the culturing to no more than 500 mOsm.

In one embodiment, the hydrolysate enrichment solution comprises a firsthydrolysate which is not derived from a plant or an animal and a secondplant-based hydrolysate. In yet another embodiment, the hydrolysatewhich is not derived from a plant or an animal and a plant-basedhydrolysate is a yeast-based hydrolysate. In still another embodiment,the plant-based hydrolysate is a soy-based hydrolysate. In oneembodiment, the hydrolysate enrichment solution consists essentially ofabout 50-280 g/kg, e.g., 250 to 280 g/kg, of a soy-based hydrolysate andabout 75-300 g/kg, e.g., 150 to 180 g/kg, of a yeast-based hydrolysate.In one embodiment, the hydrolysate enrichment solution comprises about50-280 g/kg, e.g., 250 to 280 g/kg, of a soy-based hydrolysate and about75-300 g/kg, e.g., 150 to 180 g/kg, of a yeast-based hydrolysate.

In one embodiment, the basal medium is PF CHO.

In one embodiment, the basal enrichment solution has a pH of about 9.0to 10.5.

In still another embodiment, the time period of the fed batch method isbetween about 9 to 15 days; or about 12 days.

In yet another embodiment, the basal enrichment solution is added to thecell culture production medium on at least one of the following days ofthe time period: Day 4, Day 6, Day 9, and Day 11. In one embodiment, thehydrolysate enrichment solution is added to the cell culture productionmedium on Day 4, Day 7, or Day 4 and Day 7 of the time period.

In still another embodiment, the fed batch methods further comprisesadjusting the pH of the cell culture production medium according to a pHlinear ramp, wherein the pH linear ramp comprises starting from a pH ofabout 6.5-8, e.g., 7.1 to 7.2 and resulting in a final pH of about6.5-7.0, e.g., 6.9. In one embodiment, the pH linear ramp is adjustedover a period of at least about 24 hours. In another embodiment, the pHlinear ramp is adjusted over a period of at least about 48 hours. Instill another embodiment, the pH linear ramp is adjusted over a periodof about 72 hours.

The invention also includes using the cell culture media describedherein in the fed batch method, e.g., cell culture production mediumcomprising a modified basal medium which excludes the followingcomponents sodium bicarbonate, buffer, mono-basic sodium phosphate,di-basic sodium phosphate, an osmolarity regulator, a surfactant, andmonosaccharide glucose; about 8 to 10 ml/kg or 110 to 130 mg/L ferriccitrate; about 4 to 8 mL/kg or 10 to 14 mg/kg recombinant human insulin;about 5 to 9 g/kg anhydrous glucose; about 0.1 to 1 g/kg L-glutamine;about 1 to 3 g/kg sodium bicarbonate; about 1 to 3 g/kg HEPES; about 2to 3 g/kg NaCl; about 0.1 to 2 g/kg Pluronic F-68; about 0.01 to 0.1g/kg NaH₂PO₄—H₂O; about 0.1 to 0.1 g/kg Na₂HPO₄-7H₂O; about 8 to 12 g/kgyeast-based hydrolysate; and about 6 to 8 g/kg plant-based hydrolysate.In one embodiment, the cell

cell culture production medium comprises modified basal medium; about10.0 ml/kg or 122.45 mg/L ferric citrate; about 6.0 mL/kg or 12 mg/kgrecombinant human insulin; about 7.0 g/kg anhydrous glucose; about 0.58to 0.59 g/kg L-glutamine; about 1.6 g/kg sodium bicarbonate; about 1.8g/kg HEPES; about 2.45 g/kg NaCl; about 1.0 g/kg Pluronic F-68; about0.03 to 0.04 g/kg NaH₂PO₄—H₂O; about 0.43 to 0.44 g/kg Na₂HPO₄-7H₂O;about 10.7 g/kg yeast-based hydrolysate; and about 6.9 to 7.0 g/kgplant-based hydrolysate.

The invention also provides a fed batch method of producing an anti-IL12antibody, such as, for example, a fully human anti-IL12 antibody (e.g.,ABT-874) comprising culturing CHO cells comprising a nucleic acidencoding the antibody in a cell culture comprising a cell cultureproduction medium, feeding the CHO cells by adding a hydrolysateenrichment solution and a basal enrichment solution to the cell cultureduring a time period, wherein the basal enrichment solution comprises abasal medium, asparagine, and glucose, and wherein the hydrolysateenrichment solution comprises at least two different non-animal-basedhydrosylates, such that the anti-IL12 antibody is produced.

In one embodiment, the hydrolysate enrichment solution further comprisesglucose.

In one embodiment, the invention also includes recovering the anti-IL12antibody.

In one embodiment, the cell culture is cultured at a temperature rangingfrom about 32 to 38° C., e.g., about 33° C.

In one embodiment of the invention, the cell culture production mediumis maintained at between 20-65% dissolved oxygen, e.g., at about 40%dissolved oxygen.

In yet another embodiment, the cell culture production medium has a pHof about 6.7 to 7.2.

In a further embodiment of the invention, the hydrolysate enrichmentsolution comprises a hydrolysate which is not derived from a plant or ananimal and a plant-based hydrolysate. In one embodiment, the hydrolysatewhich is not derived from a plant or an animal is a yeast-basedhydrolysate. In another embodiment, the plant-based hydrolysate is asoy-based hydrolysate. In still another embodiment, the hydrolysateenrichment solution consists essentially of about 50-225 g/kg, e.g., 150to 180 g/kg, of a soy-based hydrolysate; about 75-300, e.g., 250 to 280g/kg of a yeast-based hydrolysate; and about 1-5 g/L, e.g., 2 to 3 g/L,of glucose. In still another embodiment, the hydrolysate enrichmentsolution comprises about 50-225 g/kg, e.g., 150 to 180 g/kg, of asoy-based hydrolysate, about 75-300, e.g., 250 to 280 g/kg of ayeast-based hydrolysate, and about 1-5 g/L, e.g., 2 to 3 g/L, ofglucose. In one embodiment, the basal enrichment solution comprises abasal medium, asparagine, and glucose.

In yet another embodiment, the basal enrichment solution has a pH ofabout 9-10, e.g., about 9.7, and an osmolarity of about 1400 to 1500mOsm. In a further embodiment, the basal medium in the basal enrichmentsolution is PF CHO.

In one embodiment, the time period of the fed batch method is between14-15 days.

In one embodiment, the basal enrichment solution is added to the cellculture production medium every other day beginning on day 5 of the timeperiod.

In one embodiment of the invention, the hydrolysate enrichment solutionis added to the cell culture production medium every day beginning onday 6 of the time period. In still another embodiment, the basalenrichment solution and the hydrolysate enrichment solution are added tothe cell culture production medium every day beginning on day 5 of thetime period.

The invention also includes using the cell culture media describedherein in the fed batch method, e.g., cell culture production mediumcomprising a modified basal medium excluding the following componentssodium bicarbonate, buffer, mono-basic sodium phosphate, di-basic sodiumphosphate, an osmolarity regulator, a surfactant, and monosaccharideglucose; about 8 to 12 ml/kg or 110 to 130 mg/L ferric citrate; about 5to 8 mL/kg or 11 to 15 mg/kg recombinant human insulin; about 5 to 9g/kg anhydrous glucose; about 0.1 to 1 g/kg L-glutamine; about 1 to 2g/kg sodium bicarbonate about 1 to 2 g/kg HEPES; about 2 to 3 g/kg NaCl;about 0.1 to 2 g/kg Pluronic F-68; about 0.01 to 0.1 g/kg NaH₂PO₄—H₂O;about 0.1 to 1 g/kg Na₂HPO₄-7H₂O; about 6 to 12 g/kg yeast-basedhydrolysate; and about 6 to 8 g/kg plant-based hydrolysate. In oneembodiment, the cell culture production medium comprises about 10 ml/kgor 122.45 mg/L ferric citrate; about 6.5 mL/kg or 13 mg/kg recombinanthuman insulin; about 7.0 g/kg anhydrous glucose; about 0.58 to 0.59 g/kgL-glutamine; about 1.6 g/kg sodium bicarbonate; about 1.8 g/kg HEPES;about 2.45 g/kg NaCl; about 1.0 g/kg Pluronic F-68; about 0.03 to 0.04g/kg NaH₂PO₄—H₂O; about 0.43 to 0.44 g/kg Na₂HPO₄-7H₂O; about 10.7 g/kgyeast-based hydrolysate; and about 6.9 to 7.0 g/kg plant-basedhydrolysate.

In one embodiment, the invention features methods for culturing cells ona large scale. In one embodiment, the large scale cell culture isgreater than about 10 L. In another embodiment, the large scale cellculture is about 13 L.

The invention also provides combination feed solutions which areadvantageous because these solutions provide a combination of nutrientsin one solution. The invention includes a combination feed solutioncomprising glucose; a basal medium; an amino acid other than glutamine;and at least two different non-animal based hydrolysates. The inventionalso includes a combination feed solution consisting essentially ofglucose; a basal medium; an amino acid other than glutamine; and atleast two different non-animal based hydrolysates.

In one embodiment, the feed solution has a pH of about 6.0 to 8.0.

In one embodiment, the combination feed solution comprises about 100 to250 g/kg glucose. In one embodiment, the combination feed solutioncomprises the amino acid asparagine, e.g., about 1.0 to 15.0 g ofasparagine; or about 3.0 to 5.0 g/kg asparagine.

In one embodiment, the at least two different non-animal basedhydrosylates in the combination feed solution are a plant-basedhydrolysate and a hydrolysate which is not animal-based or plant based.In one embodiment, the hydrolysate which is not animal-based orplant-based is a yeast-based hydrolysate. In one embodiment, theplant-based hydrolysate is a soy-based hydrolysate.

In one embodiment, the combination feed solution comprises a basalmedium that is either PF-CHO or DMEM/F12 medium. In one embodiment, thebasal cell medium is a modified basal medium and excludes the followingcomponents: sodium bicarbonate, buffer, mono-basic sodium phosphate,di-basic sodium phosphate, an osmolarity regulator, a surfactant,glutamine, and glucose.

In still another embodiment, the combination feed solution further has aturbidity of less than about 15 NTU.

The invention features a method of maintaining a steady glucose level ofa cell culture production medium comprising adding the combination feedsolutions described herein.

Another aspect of the invention is a method for making a combinationfeed solution comprising a basal medium, glucose, and at least twodifferent non-animal based hydrolysates comprising combining glucose andthe basal cell medium into a solution; adjusting the pH of the solutionof a) to about 9.5 to 10.5; adding the at least two different non-animalbased hydrolysates to the solution of b); and adjusting the pH of thesolution of c) such that the combination feed solution has a pH of about6.5 to 7.5. In one embodiment, step c) comprises adding a firsthydrolysate which is not animal-based or plant-based and a secondplant-based hydrolysate. In one embodiment, the hydrolysate which is notanimal-based or plant-based is a yeast-based hydrolysate. In stillanother embodiment, the plant-based hydrolysate is a soy-basedhydrolysate.

The invention further provides methods for increased protein, e.g.,antibody or antigen-binding portions thereof, production from mammaliancell culture. The invention provides a method for producing at leastabout 1.5 g/L of an antibody from a mammalian cell culture comprisingculturing mammalian cells comprising a nucleic acid encoding theantibody in a cell culture production medium; and adding a combinationfeed solution having a pH of about 6.7 to 7.2 to the cell cultureproduction medium, wherein the combination feed solution comprisesglucose; a basal cell medium; an amino acid other than glutamine; and atleast two different non-animal based hydrosylates, such that at leastabout 1.5 g/L of the antibody is produced. In one embodiment, at least 2g/L of the antibody is produced. In another embodiment, at least 4 g/Lof the antibody is produced. In still another embodiment, at least 5 g/Lof the antibody is produced. In a further embodiment, the inventionprovides a method for producing about 6 g/L of an antibody.

In one embodiment, the combination feed solution comprises about 100 to250 g/kg glucose.

The invention also provides a method for increasing titer of an antibodyproduced from a mammalian cell culture comprising culturing mammaliancells comprising a nucleic acid encoding the antibody in a cell cultureproduction medium; and adding a combination feed solution having a pH ofabout 6.7 to 7.2 to the cell culture production medium, wherein thecombination feed solution comprises glucose; a basal cell medium; anamino acid other than glutamine; and at least two different non-animalbased hydrolysates, such that the titer of the antibody produced is atleast 50% more than a control mammalian cell culture which is culturedaccording to steps a) and excluding step b). In one embodiment, thetiter of the antibody produced is at least 100% more than the control.In another embodiment, the titer of the antibody produced is at least150% more than the control.

In one embodiment, the combination feed solution is added when the celldensity reaches at least 2.0×10⁶ cells/mL. In one embodiment, thecombination feed solution is added when the cell density reaches atleast 3.5×10⁶ cells/ml.

The invention further provides a method for producing a protein, e.g.,an antibody or antigen-binding portion thereof, in a mammalian cellculture comprising culturing mammalian cells comprising a nucleic acidencoding the protein in a cell culture production medium; and adding acombination feed solution to the cell culture production medium using afeedback control system to monitor a metabolic indicator level in thecell culture production medium, wherein the combination feed solution isadded to the cell culture production medium at a time point determinedby the feedback control system, such that the antibody is produced. Inone embodiment, the metabolic indicator is glucose or glutamine. Inanother embodiment, the feed solution is a combination feed solutioncomprising glucose; a basal cell medium; an amino acid other thanglutamine; and at least two different non-animal based hydrolysates. Inone embodiment, the antibody is an anti-TNFα antibody, an anti-IL-12antibody, an anti-IL-18 antibody, and an anti-EPO receptor (EPO-R)antibody.

In one embodiment, a titer of at least 1.5 g/L of antibody is producedusing the methods of the invention. In another embodiment, a titer of atleast 2 g/L is produced.

In one embodiment of the invention, the combination feed solutioncomprises about 3.0 to 12.5 g/kg asparagine.

In one embodiment of the invention, the combination feed solutioncomprises about 100 to 200 g/kg glucose.

In yet another embodiment, the invention further comprises monitoring aglucose level in the cell culture medium such that the glucose level ismaintained between about 0.25 and 20.0 g/L. In one embodiment, theglucose level is monitored using an automated sampling device.

In one embodiment, the antibody, or antigen binding portion thereof,which is produced using the methods and compositions disclosed herein isselected from the group consisting of an anti-TNFα antibody, ananti-IL-18 antibody, an anti-EPO-R antibody, and an anti-IL-12 antibody.In one embodiment, the antibody, or antigen-binding portion thereof, isa fully human antibody. In one embodiment, the anti-TNFα antibody isD2E7 (adalimumab). In one embodiment, the anti-IL-18 antibody isABT-325. In one embodiment, the anti-IL-12 antibody is ABT-874.

The invention also provides a method of determining a feed profile forproducing a protein in a mammalian cell culture comprising culturingmammalian cells comprising a nucleic acid encoding the antibody in acell culture production medium; and adding a combination feed solutionto the cell culture production medium using a feedback control system tomonitor a metabolic indicator in the cell culture production medium,wherein the combination feed solution is added to the cell cultureproduction medium to meet a target metabolic indicator setpoint; anddetermining the amount of the combination feed solution added to thecell culture production medium per day, such that a feed profile isdetermined.

In one embodiment, the metabolic indicator is glucose or glutamine.

The invention also includes a fed batch method for producing a proteinin a mammalian cell culture comprising adding a combination feedsolution to the mammalian cell culture according to the feed profiledetermined using the methods of the invention.

Another aspect of the invention is improved cell culture media whichinclude sodium butyrate and/or N-acetylcysteine. The invention featuresa method of producing an antibody in a mammalian cell culture such thatthe titer of the antibody is at least 300 mg/L, said method comprisingculturing mammalian cells comprising a nucleic acid encoding theantibody in a cell culture production medium; adding sodium butyrate,N-acetylcysteine, or a combination thereof, to the cell culture medium,wherein the sodium butyrate is added to a final concentration of about0.1 mM to 10 mM and the N-acetylcysteine is added to a finalconcentration of about 1 mM to 80 mM, such that the antibody is producedat a titer of at least 300 mg/L. In one embodiment, the antibody titeris at least about 100 mg/L. In one embodiment, the antibody titer is atleast about 200 mg/L. In one embodiment, the antibody titer is at leastabout 250 mg/L. In one embodiment, the antibody titer is at least about300 mg/L. In one embodiment, the antibody titer is at least about 400mg/L.

The invention also provides a method of producing an antibody in amammalian cell culture such that the titer of the antibody is at least10% greater than a control mammalian cell culture, said methodcomprising a) culturing mammalian cells comprising a nucleic acidencoding the antibody in a cell culture production medium; and b) addingsodium butyrate, N-acetylcysteine, or a combination thereof, to the cellculture medium, wherein the sodium butyrate is added to a finalconcentration of about 0.1 mM to 10 mM and the N-acetylcysteine is addedto a final concentration of about 1 mM to 80 mM, such that the titer ofthe antibody is at least 10% greater than the control, wherein thecontrol mammalian cell culture comprises step a) and excludes step b).In one embodiment, the antibody titer of the mammalian cell culture isimproved at least 29% over the control mammalian cell culture. In oneembodiment, the antibody titer of the mammalian cell culture is improvedat least 40% over the control mammalian cell culture. In one embodiment,the antibody titer of the mammalian cell culture is improved at least70% over the control mammalian cell culture. In one embodiment, theantibody titer of the mammalian cell culture is at least 90% greaterthan the control mammalian cell culture.

In one embodiment, sodium butyrate, N-acetylcysteine, or combinationthereof, is added to the mammalian cell culture during the growth phaseof the mammalian cell culture.

In one embodiment, the sodium butyrate, N-acetylcysteine, or combinationthereof is added to the mammalian cell culture between days 4 and 7 ofthe culture time.

In one embodiment, the sodium butyrate, N-acetylcysteine, or combinationthereof is added to the mammalian cell culture on day 0 of the culturetime.

In another embodiment, the final concentration of sodium butyrate isabout 0.1 mM to 10 mM. In one embodiment, the final concentration ofsodium butyrate is about 0.1 mM to 8.0 mM. In one embodiment, the finalconcentration of sodium butyrate is about 0.1 mM to 3.0 mM of sodiumbutyrate.

In one embodiment, the final concentration of N-acetylcysteine is about20 mM to 60 mM. In one embodiment, the final concentration ofN-acetylcysteine is about 10 mM. In one embodiment, the finalconcentration of N-acetylcysteine is about 8 mM.

The invention further provides a method of extending longevity of amammalian cell culture by at least 35% in comparison to a controlmammalian cell culture, said method comprising a) culturing mammaliancells comprising a nucleic acid encoding the antibody in a cell cultureproduction medium; and b) adding about 1 mM to 80 mM N-acetylcysteine tothe cell medium; a such that the longevity of the mammalian cell cultureis extended by at least 35% in comparison to a control mammalian cellculture, wherein the control mammalian cell culture comprises step a)and excludes step b).

In one embodiment, the longevity of the mammalian cell culture isextended at least about 45% in comparison to the control mammalian cellculture. In one embodiment, the longevity of the mammalian cell cultureis extended at least about 55% in comparison to the control mammaliancell culture.

In one embodiment, the method of the invention features adding a finalconcentration of about 8 mM N-acetylcysteine to the cell cultureproduction medium.

In one embodiment, the antibody, or antigen-binding portion thereof, isselected from the group consisting of an anti-TNFα antibody, ananti-IL-18 antibody (e.g., ABT-325), and an anti-IL-12 antibody.

The invention provides a serum-free cell culture medium comprising PartA, Part B, and Part C, wherein Part A consists essentially of a modifiedbasal medium which excludes the following components: sodiumbicarbonate, a buffer, mono-basic sodium phosphate, di-basic sodiumphosphate, an osmolarity regulator, a surfactant, and monosaccharideglucose; Part B consists essentially of an inorganic iron source; andPart C comprises a recombinant growth factor; a buffer; an osmolarityregulator; an energy source; and at least two different non-animalhydrolysates. In one embodiment, Part C consists essentially of arecombinant growth factor; a buffer; an osmolarity regulator; an energysource; and at least two different non-animal hydrolysates

The invention also provides a serum-free cell culture production mediumcomprising a modified basal medium having reduced vitamin content andexcluding the following components sodium bicarbonate, buffer,mono-basic sodium phosphate, di-basic sodium phosphate, an osmolarityregulator, a surfactant, and monosaccharide glucose; about 10 ml/kg or122.45 mg/L ferric citrate; about 6.5 mL/kg or 13 mg/kg recombinanthuman insulin; about 7.0 g/kg anhydrous glucose; about 0.58 to 0.59 g/kgL-glutamine; about 1.6 g/kg sodium bicarbonate about 1.8 g/kg HEPES;about 2.45 g/kg NaCl; about 1.0 g/kg Pluronic F-68; about 0.03 to 0.04g/kg NaH₂PO₄.H₂O; about 0.43 to 0.44 g/kg Na₂HPO₄.7H₂O; about 10.7 g/kgyeast-based hydrolysate; and about 6.9 to 7.0 g/kg plant-basedhydrolysate.

The invention also provides a serum-free cell culture medium comprising:a modified basal medium, having reduced vitamin content and excludingthe following components sodium bicarbonate, buffer, mono-basic sodiumphosphate, di-basic sodium phosphate, an osmolarity regulator, asurfactant, and monosaccharide glucose; about 150 g/kg anhydrousglucose; about 5.0 g/kg L-asparagine monohydrate; about 1.6 g/kg sodiumbicarbonate; about 65 g/kg yeast-based hydrolysate; and about 41 g/kgplant-based hydrolysate.

The invention further provides a serum-free cell culture mediumcomprising: a modified basal medium having reduced vitamin content andexcluding the following components sodium bicarbonate, buffer,mono-basic sodium phosphate, di-basic sodium phosphate, an osmolarityregulator, a surfactant, and monosaccharide glucose; about 10 ml/kg or122.45 mg/L ferric citrate; about 6.5 mL/kg or 13 mg/kg recombinanthuman insulin; about 200 g/kg anhydrous glucose; about 0.58 to 0.59 g/kgL-glutamine; about 1.6 g/kg sodium bicarbonate about 1.8 g/kg HEPES;about 2.45 g/kg NaCl; about 1.0 g/kg Pluronic F-68; about 0.03 to 0.04g/kg NaH₂PO₄.H₂O; about 0.43 to 0.44 g/kg Na₂HPO₄.7H₂O; about 10.7 g/kgyeast-based hydrolysate; and about 6.9 to 7.0 g/kg plant-basedhydrolysate.

The invention also provides the following embodiments for improvedmedia. The invention includes an improved media for culturing CHO cellsto express recombinant biologics comprising parts A, B and C wherein:Part A comprises water, amino acids, vitamins and other co-factors; PartB comprises an inorganic iron source; and Part C comprises recombinantgrowth factors, buffers, an osmolarity regulator, an energy source,non-ferrous metal ions, hydrolysates and additional agents.

In one embodiment, part C comprises sodium bicarbonate, HEPES, mono- anddi-basic sodium phosphates, sodium chloride, Pluronic F-68 and Glucose.In another embodiment, 1.5 g/L of sodium bicarbonate is added. In afurther embodiment, 1.8 g/L HEPES is added. In still another embodiment,0.1-0.5 g/L mono- and di-basic sodium phosphates is added. In yetanother embodiment, 1 g/L to 6.5 g/L sodium chloride is added. In afurther embodiment, 1.0 g/L Pluronic F-68 is added. In one embodiment, 1g/L to 7 g/L glucose is added. In one embodiment, the vitamins areselected from the group consisting of PABA (p-aminobenzoic acid),Biotin, D-Ca Pantothenate (vitamin B5), Folic acid, i-Inositol,Niacinamide, Pyrodoxine (vitamin B6), Riboflavin (vitamin B2), Thiamine(vitamin B1), and Cyanocobalamin (vitamin B12). In another embodiment,the other co-factors are selected from the group consisting of lipidfactors, an alcohol amine, amino acids and peptides. In still anotherembodiment, the lipid factors are selected from the group consisting ofcholine chloride and phosphatidylcholine. In yet another embodiment, thealcohol amine is ethanolamine. In one embodiment, the amino acids areselected from the group consisting of asparagine, glutamine andputrescine.

In one embodiment, the peptide is glutathione. In one embodiment, 0.4mg/L to 1.65 mg/L glutathione is added.

In still another embodiment, the inorganic iron source in Part B isferric citrate. In one embodiment, 10 mL/L or 122 mg/L ferric citrate isadded. In yet another embodiment, the ferric citrate is held to aconcentration of 122 mg/L. In one embodiment, the recombinant growthfactor is insulin or a recombinant analog, IGF-1 or a combination ofinsulin and IGF-1. In one embodiment, 4 mg/L to 13 mg/L insulin or arecombinant analog is added. In another embodiment, 25 ng/L to 150 ng/LIGF-1 is added. In yet another embodiment, 50 ng/L to 100 ng/L IGF-1 isadded. In still another embodiment, 25 ng/L to 150 ng/L IGF-1 issupplemented to the insulin. In one embodiment, 50 ng/L to 100 ng/LIGF-1 is supplemented to the insulin.

In still another embodiment, the osmolarity regulator is selected from agroup consisting of NaCl, KCl, KNO₃. In one embodiment, 0 g/L to 10 g/Losmolarity regulator is added. In another embodiment, 0 g/L to 6.5 g/Losmolarity regulator is added.

In still another embodiment, the energy source is a monosaccharide,e.g., glucose (e.g., D-glucose), maltose, mannose, galactose andfructose. In one embodiment, 1.0 to 7.0 g/L glucose is added. In anotherembodiment, 1.5 to 5.0 g/L glucose is added.

In yet another embodiment, the non-ferrous metal ions are added in theform of chloride and sulfate salts. In one embodiment, the non-ferrousmetal ions are selected from the group consisting of potassium,magnesium, cupric, selenium, zinc, nickel, manganese, tin, cadmium,molybdate, vanadate and silicate. In one embodiment, the buffer isselected from the group consisting of carbonates, chlorides, sulphatesand phosphates. In one embodiment, the buffer is selected from the groupconsisting of NaHCO₃, CaCl₂, MGSO₄, NaH₂PO₄, Na₂HPO₄, C₃H₃O₃Na andN-[2-hydroxyethyl]piperazine-N′-[2-ethansul-phonic acid] known as HEPES.

In one embodiment of the invention, one of the additional agents addedto the medium is methotrexate. In one embodiment, the methotrexate isused for growing CHO cells that express anti-IL-18, anti-IL12,anti-TNF-alpha (e.g., fully human anti-TNF alpha), or anti-EPO-Rantibodies. In one embodiment, 100 nM to 5000 nM, is added. In oneembodiment, 500 nM methotrexate is added to the medium. In oneembodiment, 100 nM methotrexate is added. In one embodiment, 5000 nMmethotrexate is added.

In still another embodiment of the invention, one of the additionalagents is a cell protectorant, e.g., methyl cellulose or a pluronicpolyol (e.g., Pluronic F-68). In one embodiment, 0.5 g/L to 1.0 g/Lmethyl cellulose is added. In one embodiment, 0.5 g/L to 1.0 g/LPluronic F-68 is added. In one embodiment, 0.7 g/L to 1.2 g/L PluronicF-68 is added.

In yet another embodiment, the pH of Part A is increased to a maximum pHof 10. In one embodiment, the pH of Part A is then reduced to a minimumof 7.0 as hydrolysates are added.

The invention also provides an improved medium for CHO cells expressingfully human anti-TNF-alpha antibody comprising: 10.0 ml/kg or 122 mg/kgferric citrate; 2 mL/kg or 4.0 mg/kg recombinant human insulin; 3.5 g/kganhydrous glucose; 0.292 g/k L-glutamine; 1.6 g/kg Sodium bicarbonate;0.031 g/kg NaH₂PO₄—H₂O; 0.436 g/kg of Na₂HPO₄-7H₂O; 2.0 g/kgHydrolysate; and 2.50 mL/kg Methotrexate.

The invention also provides an improved medium for CHO cells expressingfully human anti-TNF-alpha antibody comprising: 10.0 ml/kg or 122 mg/kgferric citrate; 6.0 mL/kg or 12 mg/kg recombinant human insulin; 7.0g/kg anhydrous glucose; 0.584 g/kg L-glutamine; 1.6 g/kg sodiumbicarbonate; 1.8 g/kg HEPES; 2.45 g/kg NaCl; 1.0 g/kg Pluronic F-68;0.031 g/kg NaH₂PO₄—H₂O; 0.436 g/kg Na₂HPO₄-7H₂O; 10.7 g/kg hydrolysate;6.92 g/kg Phytone peptone; and 2.50 mL/kg Methotrexate.

Also included in the invention is an improved medium for CHO cellsexpressing fully human anti-TNF-alpha antibody comprising: 10 ml/kg or122 mg/L ferric citrate; 3.88 mL/kg or 7.8 mg/kg recombinant humaninsulin; 7.0 g/kg anhydrous glucose; 0.876 g/kg L-glutamine; 0.45 g/kgL-asparagine monohydrate; 1.6 g/kg sodium bicarbonate; 1.8 g/kg HEPES;2.67 g/kg NaCl; 1.0 g/k Pluronic F-68; 0.031 g/kg NaH₂PO₄—H₂O; 0.436g/kg Na₂HPO₄-7H₂O; 10.7 g/kg Hydrolysate; 6.92 g/kg Phytone peptone; and2.50 mL/kg Methotrexate.

The invention further provides an improved medium for CHO cellsexpressing fully human anti-TNF-alpha antibody comprising: 10 ml/kg or122 mg/L ferric citrate; 3.88 mL/kg or 7.8 mg/kg recombinant humaninsulin; 7.0 g/kg anhydrous glucose; 0.876 g/kg L-glutamine; 0.45 g/kgL-asparagine monohydrate; 1.6 g/kg sodium bicarbonate; 1 .g/kg HEPES;2.67 g/kg NaCl; 1.0 g/k Pluronic F-68; 0.031 g/kg NaH₂PO₄—H₂O; 0.436g/kg Na₂HPO₄-7H₂O; 4.0 g/kg Hydrolysate; 2.6 g/kg Phytone peptone; and2.50 mL/kg Methotrexate.

Another aspect of the invention is an improved medium for CHO cellsexpressing an anti-IL-18 antibody comprising: 10 ml/kg or 122 mg/Lferric citrate; 3.88 mL/kg or 7.8 mg/kg recombinant human insulin; 7.0g/kg anhydrous glucose; 0.876 g/kg L-glutamine; 0.45 g/kg L-asparaginemonohydrate; 1.6 g/kg sodium bicarbonate; 1.8 g/kg HEPES; 2.675 g/kgNaCl; 1.0 g/k Pluronic F-68; 0.031 g/kg NaH₂PO₄—H₂O; 0.436 g/kgNa₂HPO₄-7H₂O; 4.0 g/kg yeast source hydrolysate; 2.579 g/kg Phytonepeptone; and 2.50 mL/kg Methotrexate.

The invention provides an improved medium for CHO cells expressing ananti-IL-18 antibody comprising: 10 ml/kg or 122 mg/L ferric citrate; 6.5mL/kg or 13 mg/kg recombinant human insulin; 7.0 g/kg anhydrous glucose;0.584 g/kg L-glutamine; 1.6 g/kg sodium bicarbonate; 1.8 g/kg HEPES;2.45 g/kg NaCl; 1.0 g/k Pluronic F-68; 0.031 g/kg NaH₂PO₄—H₂O; 0.436g/kg Na₂HPO₄-7H₂O; 10.7 g/kg yeast hydrolysate; and 6.92 g/kg Phytonepeptone.

The invention provides an improved medium for CHO cells expressing ananti-IL-18 antibody comprising: 150.0 g/kg anhydrous glucose; 5.0 g/kgL-asparagine monohydrate; 65.0 g/kg yeast hydrolysate; and 41.0 g/kgPhytone peptone.

The invention also provides an improved medium for CHO cells expressingan anti-IL-18 antibody comprising: 10 ml/kg or 122 mg/kg ferric citrate;6.5 mL/kg or 13 mg/kg recombinant human insulin; 200.0 g/kg anhydrousglucose; 0.584 g/kg L-glutamine; 1.6 g/kg sodium bicarbonate 1.8 g/kgHEPES; 2.45 g/kg NaCl; 1.0 ml/kg Pluronic F-68; 0.031 g/kg NaH₂PO₄—H₂O;0.436 g/kg Na₂HPO₄-7H₂O; 10.7 g/kg yeast hydrolysate; and 6.92 g/kgPhytone peptone.

The invention includes an improved medium for CHO cells expressing ananti-IL-18 antibody comprising: 10 ml/kg or 122 mg/kg ferric citrate; 2mL/kg or 4 mg/kg recombinant human insulin; 3.5+1.5 g/kg anhydrousglucose; 0.292 g/kg L-glutamine; 1.6 g/kg sodium bicarbonate; 2 g/kgyeast hydrolysate; and 0.25 mL/kg methotrexate.

The invention includes an improved medium for CHO cells expressing ananti-IL-18 antibody comprising: 10 ml/kg or 122 mg/kg ferric citrate; 2mL/kg or 4 mg/kg recombinant human insulin; 3.5+1.5 g/kg anhydrousglucose; 0.292 g/kg L-glutamine; 1.6 g/kg sodium bicarbonate; 11 g/kgyeast hydrolysate; and 0.250 mL/kg methotrexate.

The invention includes an improved medium for CHO cells expressing ananti-IL-18 antibody comprising: 10 ml/kg or 122 mg/kg ferric citrate; 2mL/kg or 4 mg/kg recombinant human insulin; 200 g/l anhydrous glucose;0.292 g/kg L-glutamine; 1.6 g/kg sodium bicarbonate; 8 g/kg yeasthydrolysate; and 0.250 mL/kg methotrexate.

The invention includes an improved medium for CHO cells expressing ananti-IL-18 antibody comprising: 10 ml/kg or 122 mg/kg ferric citrate;3.88 mL/kg or 7.76 mg/L recombinant human insulin; 7.0 g/l anhydrousdextrose; 0.876 g/L L-glutamine; 1.6 g/kg sodium bicarbonate; 1.8 g/LHEPES; 2.67 g/L NaCl; 1.0 g/L Pluronic; 0.031 g/L NaH₂PO4.H₂O; 0.436 g/LNa₂HPO4.7H₂O; 4.0 g/L yeastolate; 2.579 g/L phytone peptone; 0.05 mL/kgmethotrexate; 3.5 mL/L 2N NaOH; and 2.91 g/L 2N HCl; which results in afinal pH of 7.10 to 7.20 and a final osmolality of 373 to 403 mOsmo/kg.

The invention includes an improved media for CHO cells expressing ananti-IL-18 antibody comprising: 10 ml/kg or 122 mg/kg ferric citrate; 13mg/L recombinant human insulin; 7.0 g/l anhydrous dextrose; 0.584 g/LL-glutamine; 1.6 g/kg sodium bicarbonate; 1.8 g/L HEPES; 2.45 g/L NaCl;1.0 g/L Pluronic; 0.031 g/L NaH₂PO₄.H₂O; 0.436 g/L Na₂HPO₄.7H₂O; 10.7g/L yeastolate; 6.92 g/L phytone peptone; 0.05 mL/kg methotrexate; 5.67mL/L 2N NaOH; and 2.5 g/L 2N HCl; which results in a final pH of 7.10 to7.20 and a final osmolality of 373 to 403 mOsmo/kg.

The invention includes an improved medium for CHO cells expressing ananti-IL-18 antibody comprising: 10 ml/kg or 122 mg/kg ferric citrate; 4mg/kg recombinant human insulin; 1.5 g/kg anhydrous dextrose; 0.292 g/kgL-glutamine; 1.6 g/kg sodium bicarbonate; 2.0 g/L yeastolate; and 0.25mL/kg methotrexate; which results in a final pH of 7.10 to 7.30 and afinal osmolality of 300 to 340 mOsmo/kg.

The invention includes an improved medium for CHO cells expressing ananti-IL-18 antibody comprising: 10 ml/kg or 122 mg/kg ferric citrate; 13mg/kg recombinant human insulin; 7.0 g/kg anhydrous dextrose; 0.584 g/kgL-glutamine; 1.6 g/kg sodium bicarbonate; 1.8 g/kg HEPES; 2.45 g/kgNaCl; 1.0 g/kg Pluronic F-68; 0.031 g/kg NaH₂PO4.H₂O; 0.436 g/kgNa₂HPO₄.7H₂O; 10.7 g/L yeastolate; and 6.92 g/kg phytone peptone; n5.67mL/kg NaOH; and 2.5 mL/kg HCl; which results in a final pH of 7.10 to7.20 and a final osmolality of 373 to 403 mOsmo/kg.

The invention includes an improved medium for CHO cells expressing ananti-IL-18 antibody comprising: 10 ml/kg or 122 mg/kg ferric citrate;7.76 mg/kg recombinant human insulin; 7.0 g/l anhydrous dextrose; 0.876g/kg L-glutamine; 1.6 g/kg sodium bicarbonate; 1.8 g/kg HEPES; 2.67 g/kgNaCl; 1.0 g/kg Pluronic F-68; 0.031 g/kg NaH₂PO₄.H₂O; 0.436 g/kgNa₂HPO₄.7H₂O; 4.0 g/L yeastolate; and 2.579 g/L phytone peptone; 0.05mL/L methotrexate; 3.5 mL/kg NaOH; and 2.91 mL/kg HCl; which results ina final pH of 7.10 to 7.20 and a final osmolality of 373 to 403mOsmo/kg.

The invention includes an improved medium for CHO cells expressing ananti-IL-18 antibody comprising: 10 ml/kg or 122 mg/kg ferric citrate; 13mg/kg recombinant human insulin; 7.0 g/l anhydrous dextrose; 0.584 g/kgL-glutamine; 1.6 g/kg sodium bicarbonate; 1.8 g/kg HEPES; 2.45 g/kgNaCl; 1.0 g/kg Pluronic F-68; 0.031 g/kg NaH₂PO4.H₂O; 0.436 g/kgNa₂HPO₄.7H₂O; 10.7 g/L yeastolate; and 6.92 g/L phytone peptone; 0.05mL/L methotrexate; 5.67 mL/kg NaOH; and 2.5 mL/kg HCl; which results ina final pH of 7.10 to 7.20 and a final osmolality of 373 to 403mOsmo/kg.

The invention includes an improved medium for CHO cells expressing ananti-IL-18 antibody comprising: 10 ml/kg or 122 mg/kg ferric citrate; 13mg/kg recombinant human insulin; 7.0 g/l anhydrous dextrose; 0.584 g/kgL-glutamine; 1.6 g/kg sodium bicarbonate; 1.8 g/kg HEPES; 1.0 g/kgPluronic F-68; 0.031 g/kg NaH₂PO4.H₂O; 0.436 g/kg Na₂HPO₄.7H₂O; 14.27g/L yeastolate; 9.23 g/L phytone peptone; 0.05 mL/L methotrexate; 8.95mL/kg NaOH; and 4.1 mL/kg HCl; which results in a final pH of 7.10 to7.20 and a final osmolality of 373 to 403 mOsmo/kg.

The invention also features a method of increasing the productivity of aCHO cell line producing an IgG1 antibody by increasing the final titercomprising: adding sodium butyrate; and adding N-acetylcysteine. In oneembodiment, the IgG1 antibody is anti-IL-18. In one embodiment, theincrease in productivity is measure by an increase in the final titer.In one embodiment, the increase in the final titer is achieved throughthe addition of sodium butyrate in a concentration from 0.1 mM to 10 mM.In one embodiment, the concentration of sodium butyrate is 0.1 mM to 8.0mM. In one embodiment, the concentration of sodium butyrate is 0.1 mM to3.0 mM. In one embodiment, the concentration of sodium butyrate is 0.125mM to 2.0 mM. In one embodiment, the concentration of sodium butyrate is0.125 mM. In one embodiment, the increase in final titer is 10-80%. Inone embodiment, the increase in final titer is 20-60%. In oneembodiment, the increase in final titer is 35-55%. In one embodiment,the increase in final titer is 40%. In one embodiment, improved cellculture longevity is achieved through the addition of N-acetylcysteinein a concentration from 0.1 mM to 10 mM. In one embodiment, the increasein cell culture longevity is 5-50%.

The invention also provides a cell culture medium increasing theproductivity of a CHO cell line producing an IgG1 antibody by increasingthe final titer comprising: SR-371; and sodium butyrate. In oneembodiment, the IgG1 antibody is anti-IL-18. In one embodiment, theproductivity increase is measured by the final anti IL-18 titerincreasing by 10-80%. In one embodiment, the productivity increase ismeasured by the final anti IL-18 titer increasing by 20-60%. In oneembodiment, the productivity increase is measured by the final antiIL-18 titer increasing by 35-55%. In one embodiment, the productivityincrease is measured by the final anti IL-18 titer increasing by 40%. Inone embodiment, the concentration of sodium butyrate added is 0.125 mMto 8.0 mM. In one embodiment, the concentration of sodium butyrate addedis 0.2 mM to 3.0 mM. In one embodiment, the concentration of sodiumbutyrate added is 0.3 mM to 2.0 mM. In one embodiment, the concentrationof sodium butyrate added is 0.125 mM. In one embodiment, theconcentration of N-acetylcysteine added is 1 mM to 10 mM. In oneembodiment, the concentration of N-acetylcysteine added is 5 mM to 10mM. In one embodiment, the average final titer is increased by 5-50%. Inone embodiment, the average final titer is increased by 15-35%. In oneembodiment, the average final titer is increased by 25-35%.

In one embodiment, the human neutralizing, antibody, or anantigen-binding portion thereof, having a high affinity for human TNFαis an isolated human antibody, or an antigen-binding portion thereof,with a light chain variable region (LCVR) having a CDR3 domaincomprising the amino acid sequence of SEQ ID NO:3, or modified from SEQID NO:3 by a single alanine substitution at position 1, 4, 5, 7 or 8,and with a heavy chain variable region (HCVR) having a CDR3 domaincomprising the amino acid sequence of SEQ ID NO:4, or modified from SEQID NO:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8,9, 10 or 11. Preferably, the LCVR further has a CDR2 domain comprisingthe amino acid sequence of SEQ ID NO:5 (i.e., the D2E7 VL CDR2) and theHCVR further has a CDR2 domain comprising the amino acid sequence of SEQID NO: 6 (i.e., the D2E7 VH CDR2). Even more preferably, the LCVRfurther has CDR1 domain comprising the amino acid sequence of SEQ IDNO:7 (i.e., the D2E7 VL CDR1) and the HCVR has a CDR1 domain comprisingthe amino acid sequence of SEQ ID NO:8 (i.e., the D2E7 VH CDR1).

In another embodiment, the human neutralizing, antibody, or anantigen-binding portion thereof, having a high affinity for human TNFαis an isolated human antibody, or an antigen-binding portion thereof,with a light chain variable region (LCVR) comprising the amino acidsequence of SEQ ID NO:1 (i.e., the D2E7 VL) and a heavy chain variableregion (HCVR) comprising the amino acid sequence of SEQ ID NO:2 (i.e.,the D2E7 VH). In certain embodiments, the antibody comprises a heavychain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgMor IgD constant region. Preferably, the heavy chain constant region isan IgG1 heavy chain constant region or an IgG4 heavy chain constantregion. Furthermore, the antibody can comprise a light chain constantregion, either a kappa light chain constant region or a lambda lightchain constant region. Preferably, the antibody comprises a kappa lightchain constant region.

DESCRIPTION OF FIGURE

FIG. 1 graphically depicts ABT-874 growth titer as a function of viablecell density at seed. Titer results at day 15 are strongly correlated tothe viable cell density at feed. A polynomial fit to the above datasuggests the optimal feed density to occur around 3.5·10⁶ cells·ml⁻¹.Process parameters were pH=6.9, T=35° C., DO=40%, inoculum ratio 1:5 or1:4 into 4× medium, feeding begun at specified density, 1% of initialvolume for 10 days

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

While the terminology used in this application is standard within theart, definitions of certain terms are provided herein to assure clarityand definiteness to the meaning of the claims.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or VL) and a light chain constant region.The light chain constant region is comprised of one domain, CL. The VHand VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Examples of antibodies which maybe produced using the methods and compositions of the invention includetumor necrosis factor (TNF)-α antibodies (also referred to as anti-TNFαantibodies), interleukin (IL)-12 antibodies (also referred to asanti-IL12 antibodies), interleukin (IL)-18 antibodies (also referred toas anti-IL18 antibodies), and EPO/R antibodies (also referred to hereinas anti-EPO/R antibodies). TNFα antibodies which may be produced usingthe invention are described in further detail in U.S. Pat. Nos.6,090,382; 6,258,562; and 6,509,015, each of which is incorporatedherein by reference in its entirety.

The invention may also be used to produce antibody fragments. The term“antigen-binding portion” or “antigen binding fragment” of an antibody(or simply “antibody portion”), as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., hTNFα). It has been shown that fragments of afull-length antibody can perform the antigen-binding function of anantibody. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al. (1989) Nature 341:544-546), which consists of a VH or VLdomain; (vi) an isolated complementarity determining region (CDR); and(vii) a dual variable domain (DVD) antibody. Furthermore, although thetwo domains of the Fv fragment, VL and VH, are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair to form monovalent molecules (known as singlechain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; andHuston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Suchsingle chain antibodies are also encompassed within the term“antigen-binding portion” of an antibody. Other forms of single chainantibodies, such as diabodies are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites (see e.g., Holliger et al. (1993)Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure2:1121-1123). Examples of antibody portions which may be produced by themethods of the invention are described in further detail in U.S. Pat.Nos. 6,090,382, 6,258,562, 6,509,015, each of which is incorporatedherein by reference in its entirety. Production of antibody fragments orportions using the methods and compositions of the invention are alsoincluded within scope of the invention.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell, antibodiesisolated from a recombinant, combinatorial human antibody library(described further below), antibodies isolated from an animal (e.g., amouse) that is transgenic for human immunoglobulin genes (see e.g.,Taylor et al. (1992) Nucl. Acids Res. 20:6287) or antibodies prepared,expressed, created or isolated by any other means that involves splicingof human immunoglobulin gene sequences to other DNA sequences. Suchrecombinant human antibodies have variable and constant regions derivedfrom human germ line immunoglobulin sequences. In certain embodiments,however, such recombinant human antibodies are subjected to in vitromutagenesis (or, when an animal transgenic for human Ig sequences isused, in vivo somatic mutagenesis) and thus the amino acid sequences ofthe VH and VL regions of the recombinant antibodies are sequences that,while derived from and related to human germ line VH and VL sequences,may not naturally exist within the human antibody germ line repertoirein vivo.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hTNFα is substantially free of antibodies that specifically bindantigens other than hTNFα). An isolated antibody that specifically bindshTNFα may, however, have cross-reactivity to other antigens, such asTNFα molecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The term “basal medium” refers to any medium which is capable ofsupporting growth of cells. The basal medium supplies standard inorganicsalts, such as zinc, iron, magnesium, calcium and potassium, as well astrace elements, vitamins, an energy source, a buffer system, andessential amino acids. Examples of basal media include, but are notlimited to, Dulbecco's Modified Eagle's Medium (DMEM), DME/F12, MinimalEssential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12,.α-Minimal Essential Medium (α-MEM), Glasgow's Minimal Essential Medium(G-MEM), PF CHO (SAFC Biosciences) and Iscove's Modified Dulbecco'sMedium.

The term “modified basal medium,” as used herein, refers to a basalmedium from which at least one standard ingredient, component, ornutrient, (i.e., at least one ingredient, component, or nutrient foundin a classically formulated basal medium known in the art), has beenexcluded, decreased, or increased. The term “modified” as used in thecontext of “modified basal medium” may also refer to changes inproportion between the individual components within the basal medium. Ina preferred embodiment of the invention, a modified basal mediumexcludes at least one of the following components: sodium bicarbonate, abuffer, sodium phosphate (mono-based and/or di-basic), an osmolarityregulator, a surfactant, and glucose, e.g., monosaccharide glucose.

As used herein, the terms “cell culture medium,” “culture medium,” and“medium formulation” refer to a nutritive solution for the maintenance,growth, propagation, or expansion of cells in an artificial in vitroenvironment outside of a multicellular organism or tissue. Cell culturemedium may be optimized for a specific cell culture use, including, forexample, cell culture growth medium which is formulated to promotecellular growth, or cell culture production medium which is formulatedto promote recombinant protein production. The terms nutrient,ingredient, and component are used interchangeably herein to refer tothe constituents that make up a cell culture medium.

The terms “cell culture production medium” or “production medium” asused herein refer to cell culture medium designed to be used during theproduction phase of a cell culture. In a preferred embodiment,production medium is designed for recombinant protein expression duringproduction phase. Examples of production media are provided herein,including Tables 2-7 of the Examples section.

The terms “fed batch cell culture” and “fed batch culture,” as usedherein, refer to a cell culture wherein the cells, preferably mammalian,and culture medium are supplied to the culturing vessel initially andadditional culture nutrients are fed, continuously or in discreteincrements, to the culture during culturing, with or without periodiccell and/or product harvest before termination of culture.

A “fed batch method,” refers to a method by which a fed batch cellculture is supplied with additional nutrients. For example, a fed batchmethod may comprise adding supplemental media according to a determinedfeeding schedule within a given time period.

As used herein, the term “feed” refers to any addition of any substancemade to a culture after inoculation. Feeding can be one or moreadditions.

As used herein, the terms “feed solution,” “feed medium” and “feedingmedium” refer to a medium containing one or more nutrients that is addedto the culture beginning at some time after inoculation. In oneembodiment, the feed solution is a combination feed comprising a basalmedium and at least one hydrolysate, e.g., soy-based, hydrolysate, ayeast-based hydrolysate, or a combination of the two types ofhydrolysates. In another embodiment of the invention, a feed solutionmay include only a basal medium, such as a concentrated basal medium, ormay include only hydrolysates, or concentrated hydrolysates.

As used herein, the term “feedback control system” refers to a processof monitoring a given parameter, whereby an additional agent is added oran environmental modification of the cell culture is performed in orderto meet a desired parameter setpoint. In one embodiment, the givenparameter is the glucose level of a mammalian cell culture, whereby theglucose level is used to determine when a feed solution, e.g., acombination feed solution, should be added to the cell culture. Afeedback control system may be used to maintain nutritional componentsneeded to optimize protein production in a mammalian cell culture.

As used herein, the term “feed profile” refers to a schedule forsupplementing a mammalian cell culture with a feed solution, e.g., acombination feed solution. A feed profile is preferable generated usinga feedback control system.

Cells may be “genetically engineered” to express a specific polypeptideor protein when recombinant nucleic acid sequences that allow expressionof the polypeptide have been introduced into the cells using methods of“genetic engineering,” such as viral infection with a recombinant virus,transfection, transformation, or electroporation. See e.g. Kaufman etal. (1990), Meth. Enzymol. 185: 487-511; Current Protocols in MolecularBiology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, andquarterly updates). Methods and vectors for genetically engineeringcells and/or cell lines to express a protein of interest are well knownto those skilled in the art. Genetic engineering techniques include butare not limited to expression vectors, targeted homologous recombinationand gene activation (see, for example, U.S. Pat. No. 5,272,071 toChappel) and trans activation by engineered transcription factors (seee.g., Segal et al., 1999, Proc. Natl. Acad. Sci. USA 96(6):2758-63).Optionally, the polypeptides are expressed under the control of aheterologous control element such as, for example, a promoter that doesnot in nature direct the production of that polypeptide. For example,the promoter can be a strong viral promoter (e.g., CMV, SV40) thatdirects the expression of a mammalian polypeptide. The host cell may ormay not normally produce the polypeptide. For example, the host cell canbe a CHO cell that has been genetically engineered to produce a humanpolypeptide, meaning that nucleic acid encoding the human polypeptidehas been introduced into the CHO cell. Alternatively, the host cell canbe a human cell that has been genetically engineered to produceincreased levels of a human polypeptide normally present only at verylow levels (e.g., by replacing the endogenous promoter with a strongviral promoter).

“Growth phase,” as used herein, refers to the period during whichcultured cells are rapidly dividing and increasing in number. Duringgrowth phase, cells may be generally cultured in a medium and underconditions designed to maximize cell proliferation.

The term “hydrolysate” includes any enzymatic digest, particularly aspecialized type of extract prepared by treating the substance to beextracted (e.g., plant components or yeast cells) with at least oneenzyme capable of breaking down the components of the substance intosimpler forms (e.g., into a preparation comprising mono- ordisaccharides and/or mono-, di- or tripeptides). An “hydrolysate” can befurther enzymatically digested, for example by papain, and/or formed byautolysis, thermolysis and/or plasmolysis. In a preferred embodiment ofthe invention, the hydrolysate is not prepared from an animal source,i.e., non-animal based. Examples of preferred non-animal basedhydrolysates include plant-based hydrolysates, e.g., a soy-basedhydrolysate, and hydrolysates which are neither derived from plant oranimal sources, e.g., a yeast-based hydrolysate.

The terms “hydrolysate enrichment solution” and “hydrolysate enrichmentmedium” refer to a medium containing a hydrolysate or a combination ofhydrolysates, i.e., hydrolysates extracted from different sources, as amain ingredient that is added to the cell culture. The hydrolysateenrichment solution may, for example, be added to the cell culture toenhance protein production. Similarly, the terms “basal enrichmentsolution” and “basal enrichment medium” refer to a medium containing abasal medium (or combination of basal media) as a main ingredient. Inone embodiment, a hydrolysate enrichment solution or a basal enrichmentsolution or a combination of the two enrichment solutions, is added to acell culture to increase productivity of a cell culture in theproduction of a protein.

The production of a protein is “increased” by the addition of anadditional agent or the alteration of a parameter of the proteinproduction process, if the amount the polypeptide produced in a culturecontaining the additional agent or altered parameter of the proteinproduction process, is more than the amount of the polypeptide producedin an otherwise identical culture that does not contain the additionalagent or altered parameter of the protein production process. Examplesof alterations to the protein production process include, but are notlimited to, addition of media supplements, increases in the amount of asupplemental media, variations in the culturing temperature, and theconcentration of oxygen at which the cells are cultured. An additionalagent may be provided to the cell culture using a supplemental solution,such as a feed solution.

The term “ingredient” refers to any compound, whether of chemical orbiological origin, that can be used in cell culture media to maintain orpromote the growth of proliferation of cells. The terms “component,”“nutrient” and ingredient” are used interchangeably and are all meant torefer to such compounds. Typical ingredients that are used in cellculture media include amino acids, salts, metals, sugars, lipids,nucleic acids, hormones, vitamins, fatty acids, proteins and the like.Other ingredients that promote or maintain cultivation of cells ex vivocan be selected by those of skill in the art within the scope of theinvention, and in accordance with the particular need.

As used herein, the term “inoculation” refers to the addition of cellsto a medium to begin the culture.

“Production phase” refers to a period during which cells are producingmaximal amounts of recombinant polypeptide or protein. A productionphase is characterized by less cell division than during a growth phase,and may also include the use of medium and culture conditions designedto maximize polypeptide production.

A “recombinant polypeptide” or “recombinant protein” is a polypeptide orprotein resulting from the process of genetic engineering. In apreferred embodiment, recombinant proteins are obtained from culturingcells expressing said proteins in a cell culture.

Transition phase” means a period of cell culture between a “growthphase” and a “production phase.” During the transition phase, the mediumand environmental conditions may be shifted from those designed tomaximize proliferation to those designed to maximize polypeptideproduction.

The present invention provides new compositions and processes for theproduction of proteins, preferably recombinant protein, e.g.,antibodies, by mammalian, e.g., Chinese Hamster Ovary (CHO), cellcultures. Cell culture media and processes described herein have beenused for recombinant protein production, particularly recombinant (fullyhuman, humanized, or chimeric) monoclonal antibody production. The mediaand processes have been modified over many antibody product lines toincorporate various improvements and advances leading to increasedgrowth and productivity of mammalian, e.g., CHO, cells. Aspects of theimproved compositions and methods of the invention are provided indetail below.

II. Proteins of Interest

Generally, the methods and compositions of the invention are useful forthe production of recombinant proteins. Recombinant proteins areproteins produced by the process of genetic engineering. Particularlypreferred proteins for production according to the methods andcompositions of the invention, are protein-based therapeutics, alsoknown as biologics. Preferably, the proteins are secreted asextracellular products.

Proteins that can be produced using the methods and compositions of theinvention include, but are not limited to, antibodies or antigen bindingfragments thereof. Numerous techniques are known in the art by which DNAencoding antibody molecules can be manipulated to yield DNAs capable ofencoding recombinant proteins such as single chain antibodies,antibodies with enhanced affinity, or other antibody-based polypeptides(see, for example, Larrick et al., 1989, Biotechnology 7:934-938;Reichmann et al., 1988, Nature 332:323-327; Roberts et al., 1987, Nature328:731-734; Verhoeyen et al., 1988, Science 239:1534-1536; Chaudhary etal., 1989, Nature 339:394-397, each of which is incorporated byreference herein). Recombinant cells producing fully human antibodies(such as are prepared using transgenic animals, and optionally furthermodified in vitro), as well as humanized antibodies, can also be used inthe invention. The term humanized antibody also encompasses single chainantibodies. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabillyet al., European Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No.4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, M.S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No.0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, European PatentNo. 0,239,400 B1; Queen et al., European Patent No. 0 451 216 B1; andPadlan, E. A. et al., EP 0 519 596 A1, each of which is incorporated byreference herein. For example, the invention can be used in theproduction of human and/or humanized antibodies that immunospecificallyrecognize specific cellular targets, e.g., any of the aforementionedproteins, the human EGF receptor, the her-2/neu antigen, the CEAantigen, Prostate Specific Membrane Antigen (PSMA), CD5, CD11a, CD18,NGF, CD20, CD45, CD52, Ep-cam, other cancer cell surface molecules,TNF-alpha, TGF-b1, VEGF, other cytokines, alpha 4 beta 7 integrin, IgEs,viral proteins (for example, cytomegalovirus). Examples of antibodieswhich can be produced using the compositions and methods of theinvention include, but are not limited to, anti-TNFα antibody, ananti-IL-12 antibody, an anti-IL-18 antibody, and an anti-EPO receptor(EPO-R) antibody. In one embodiment, the anti-TNFα antibody is a fullyhuman anti-TNFα antibody, e.g., adalimumab/D2E7 (see U.S. Pat. No.6,090,382, incorporated by reference herein; Humira®; AbbottLaboratories). In one embodiment, the anti-IL-12 antibody is a fullyhuman, anti-IL-12 antibody, e.g, ABT-874 (Abbott Laboratories; see U.S.Pat. No. 6,914,128, incorporated by reference herein). In oneembodiment, the anti-IL-18 antibody is a fully human IL-18 antibody(e.g., ABT-325), e.g. see also antibodies described in U520050147610 A1.In one embodiment, the anti-EPO/R (also referred to as ABT-007) antibodyis a fully human antibody, like that described in US Patent PublicationNo. US 20060018902 A1, hereby incorporated by reference.

Another example of the type of protein that may be produced using themethods and compositions of the invention include fusion proteins. Afusion protein is a protein, or domain or a protein (e.g. a solubleextracellular domain) fused to a heterologous protein or peptide.Examples of such fusion proteins include proteins expressed as a fusionwith a portion of an immunoglobulin molecule, proteins expressed asfusion proteins with a zipper moiety, and novel polyfunctional proteinssuch as a fusion proteins of a cytokine and a growth factor (i.e.,GM-CSF and IL-3, MGF and IL-3). WO 93/08207 and WO 96/40918 describe thepreparation of various soluble oligomeric forms of a molecule referredto as CD40L, including an immunoglobulin fusion protein and a zipperfusion protein, respectively; the techniques discussed therein areapplicable to other proteins. Another fusion protein is a recombinantTNFR:Fc, also known as entanercept. Entanercept (or Enbrel®;Amgen/Wyeth) is a dimer of two molecules of the extracellular portion ofthe p75 TNF alpha receptor, each molecule consisting of a 235 amino acidTNFR-derived polypeptide that is fused to a 232 amino acid Fc portion ofhuman IgG1. In fact, any molecule can be expressed as a fusion proteinincluding, but not limited to, the extracellular domain of a cellularreceptor molecule, an enzyme, a hormone, a cytokine, a portion of animmunoglobulin molecule, a zipper domain, and an epitope.

III. Cell Culture Media of the Invention

The present invention provides cell culture media for use in mammaliancell culture for the production or expression of recombinant proteins,e.g., antibodies or antigen-binding portions thereof. The various cellculture media described herein may be used separately or collectivelyfor improved cell culturing, including increased protein production andextended cell longevity.

In a preferred embodiment, the cell culture media of the invention isserum-free, meaning that the medium contains no serum (e.g., fetalbovine serum (FBS), horse serum, goat serum, or any other animal-derivedserum known to one skilled in the art).

In a first aspect, the invention provides a mammalian cell culturemedium which includes, in whole or in part, a modified basal medium.Modified basal cell media may be derived from standard basal cell mediaknown in the art. Suitable basal media include, but are not limited toDulbecco's Modified Eagle's Medium (DMEM), DME/F12, Minimal EssentialMedium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, α-MinimalEssential Medium (α-MEM), Glasgow's Minimal Essential Medium (G-MEM), PFCHO (see, e.g., CHO protein free medium (Sigma) or EX-CELL™ 325 PF CHOSerum-Free Medium for CHO Cells Protein-Free (SAFC Bioscience), andIscove's Modified Dulbecco's Medium. Other examples of basal media whichmay be used in the invention include BME Basal Medium (Gibco-Invitrogen;see also Eagle, H (1965) Proc. Soc. Exp. Biol. Med. 89, 36); Dulbecco'sModified Eagle Medium (DMEM, powder) (Gibco-Invitrogen (#31600); seealso Dulbecco and Freeman (1959) Virology 8, 396; Smith et al. (1960)Virology 12, 185. Tissue Culture Standards Commitee, In Vitro 6:2, 93);CMRL 1066 Medium (Gibco-Invitrogen (#11530); see also Parker R. C. et al(1957) Special Publications, N.Y. Academy of Sciences, 5, 303).

Basal medium may be modified in order to remove certain non-nutritionalcomponents found in standard basal medium, such as various inorganic andorganic buffers, surfactant(s), and sodium chloride. Removing suchcomponents from basal cell medium allows an increased concentration ofthe remaining nutritional components, and improves overall cell growthand protein expression, as described herein. In addition, omittedcomponents may be added back into the cell culture medium containing themodified basal cell medium according to the requirements of the cellculture conditions. As described below, it has been found thatseparating certain ingredients from the basal cell medium, i.e., addingmodified basal cell medium as an ingredient in a cell culture medium,and subsequently adding the ingredient back into to the cell culturemedium as a separate ingredient provides advantageous properties to thegrowth of the cell culture and protein production.

The modified basal medium of the invention excludes any, if not all, ofthe following ingredients: sodium bicarbonate, a buffer, mono-basicsodium phosphate, di-basic sodium phosphate, an osmolarity regulator, asurfactant, and monosaccharide glucose. These ingredients are commonlyfound in commercial basal cell media.

Exclusion of components, e.g., sodium bicarbonate, a buffer, mono-basicsodium phosphate, di-basic sodium phosphate, an osmolarity regulator, asurfactant, and/or monosaccharide glucose, may be done by commercialmedia services, e.g., SAFC Pharma™. One of skill in the art willappreciate that modified basal media may be obtained, in one embodiment,using a commercial cell culture media service, i.e., custom mediaservice. Examples of custom media services are provided by companiessuch as SAFC (formerly JRH Bioscience), Invitrogen, AtlantaBiologicals®, and Lonza.

Alternatively, one of ordinary skill in the art can prepare the modifiedbasal cell medium of the invention according to standard methods formaking basal cell media, wherein the specific ingredients describedherein are omitted (see, e.g., Freshney (2005) Culture of Animal Cells:A Manual of Basic Technique; BD Bionutrients Technical Manual, (2006),Third edition; Jenkins, ed. (1999), Animal Cell Biotechnology, Methodsand Protocols, Humana Press; Doyle and Griffiths, eds., (1997) EssentialTechniques: Mammalian Cell Culture, John Wiley and Sons; Butler, ed.(1991) Mammalian Cell Biotechnology: A Practical Approach OxfordUniversity Press; Darling and Morgan (1994) Animal Cells: Culture andMedia, John Wiley and Sons; Freshney, ed. (1992), Animal Cell Culture: APractical Approach (2nd ed), Oxford University Press; Pollard and Walker(1997), Basic Cell Culture Protocols (2nd Ed), Humana Press, (Part ofthe Methods in Molecular Biology series, Volume 75), each of which isincorporated by reference herein).

In one embodiment, the cell culture medium of the invention contains amodified basal cell medium, an iron source (preferably inorganic, e.g.,ferric citrate), a recombinant growth factor; a buffer; a surfactant; anosmolarity regulator; an energy source; and at least two differentnon-animal hydrolysates. In addition, the modified basal cell medium mayoptionally contain amino acids, vitamins, or a combination of both aminoacids and vitamins.

As used herein, the term “iron” means a non-animal derived source ofiron used to supplement the medium. The iron source in the cell culturemedium is preferably inorganic. The iron source is preferably inorganic,and includes, for example, ferric and ferrous salts such as ferriccitrate or ferrous sulphate. The chelated salts such as ferric citrateand ferric ammonium citrate are preferred. However, other iron sourcesmay be used which are not isolated from an animal source, for example,chemical iron chelators or recombinant protein iron carriers thatprovide equivalent amounts of iron. Iron chelate compounds which may beused include but are not limited to iron chelates ofethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(.beta.-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA),deferoxamine mesylate, dimercaptopropanol,diethylenetriamine-pentaacetic acid (DPTA), andtrans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), as wellas a ferric citrate chelate and a ferrous sulfate chelate. Aparticularly preferred source of iron is ferric citrate, which ispreferably present in the final volume of the cell culture medium in aconcentration of 0.1-1 mM. In one embodiment, the concentration offerric citrate is about 0.5 mM. In another embodiment, the concentrationof ferric citrate is 100-150 mg/L, e.g., 122 mg/L. Numbers intermediateto the above recited mM, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, and 1.0 mM, and mg/L, e.g., 100, 110, 120, 130, 140, and 150, arealso intended to be part of this invention.

Non limiting examples of growth factors that may be included in the cellculture medium, are insulin, or a recombinant analog thereof, IGF-1, anda combination of insulin and IGF-1. A particularly preferred recombinantgrowth factor is insulin, or a recombinant analog thereof, which ispreferably present in the final volume of the cell culture medium in aconcentration of between about 4 mg/L to 13 mg/L. Numbers intermediateto the above recited concentration of insulin is also intended to bepart of the invention, e.g., 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 11.5, 12, 12.5, and 13.

The cell culture medium may also include a buffer. In a preferredembodiment, a buffer is excluded from the modified basal cell medium butadded as a separate component to the cell culture medium (to which themodified basal cell medium is also added). Buffers for use in cellculture medium are known in the art. Nonlimiting examples of bufferswhich may be included the cell culture medium are phosphate buffer,HEPES, and sodium bicarbonate. In one embodiment, sodium bicarbonate isadded as a buffer to the cell culture medium at a final concentration ofabout 0.1-3 g/L. In one embodiment, sodium bicarbonate is added as abuffer to the cell culture medium at a final concentration of about 1.6g/L. In one embodiment, HEPES is added as a buffer to the cell culturemedium at a final concentration of about 0.1-3 g/L. In anotherembodiment, HEPES is added as a buffer to the cell culture medium at afinal concentration of 1.8 g/L. In one embodiment a phosphate buffer,e.g., mono- and di-basic sodium phosphates, is added to the cell culturemedium at a final concentration of between 0.01 and 0.5 g/L. Numbersintermediate to the above recited concentrations are also intended to bepart of the invention, e.g., concentration of sodium bicarbonate orHEPES 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, and 3.0 or the concentration of phosphate buffer 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14, 0.16, 0.18,0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.42,0.44, 0.46, 0.48, and 0.5 g/L.

The buffer is included to help maintain the cell culture medium at adesired pH. In one embodiment, the pH of the cell culture medium rangesfrom 6.0 to 8.0; 6.5 to 7.5; or 7.1 to 7.2. Numbers intermediate tothese pH values, e.g., 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0, as well as allother numbers recited herein, are also intended to be part of thisinvention. Ranges of values using a combination of any of the aboverecited values as upper and/or lower limits are intended to be includedin the scope of the invention.

The cell culture medium may also include an osmolarity regulator, suchas NaCl. In one embodiment, NaCl is added to the cell culture medium ata final concentration of between about 1.0 to 6.5 g/L. In oneembodiment, the osmolarity of the cell culture medium ranges from 260 to450 mOsm/kg. In one embodiment, the osmolarity of the cell culturemedium ranges from 320 to 450 mOsm/kg. Numbers intermediate to therecited NaCl concentrations and mOsm/kg values, e.g, NaCl concentrationof 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, and 6.5 or an mOsm/kgrange of 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 450, as well as numbers intermediatethereto, are also intended to be part of this invention. Ranges ofvalues using a combination of any of the above recited values as upperand/or lower limits are intended to be included in the scope of theinvention.

An energy source may also be added to the cell culture medium of theinvention. Preferably, the energy source is a monosaccharide. Examplesof monosaccharides which may be used in the cell culture medium includeglucose (e.g., D-glucose), maltose, mannose, galactose and fructose. Inone embodiment, glucose is added to the cell culture medium at a finalconcentration ranging from 3.5-7.0 g/L. In one embodiment, glucose isadded to the cell culture medium at a final concentration of no greaterthan 7.0 g/L. In one embodiment, glucose is added to the cell culturemedium at a final concentration of about 7.0 g/L. Numbers intermediateto the recited glucose concentrations, e.g., 3.5, 3.6, 3.7, 3.8, 3.9, 4,4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, and 7,as well as numbers intermediate thereto, are also intended to be part ofthis invention. Ranges of values using a combination of any of the aboverecited values as upper and/or lower limits are also intended to beincluded in the scope of the invention.

An important ingredient in the cell culture medium of the invention isthe addition of a hydrolysate. Cell culture medium of the invention mayinclude a hydrolysate derived from a single source, e.g., yeast or soy,or may include a combination of hydrolysates, e.g, yeast and soy-basedhydrolysates. Preferably, hydrolysates used in cell culture media of theinvention are non-animal based. Examples of non-animal basedhydrolysates include plant-based hydrolysates and non-plant-basedhydrolysates, e.g., yeast-based hydrolysates, Tryprone, caseinhydrolysate, yeast extract, or papain digested soy peptone. Hydrolysatesused in the media of the invention are commercially available,including, for example, HyPep 1510®, Hy-Soy®, Hy-Yeast 412® and Hi-Yeast444®, from sources such as Quest International, Norwich, N.Y.,OrganoTechnie, S.A. France, Deutsche Hefewerke GmbH, Germany, or DMVIntl. Delhi, N.Y. Sources of yeast extracts and soy hydrolysates arealso disclosed in WO 98/15614, WO 00/03000, WO 01/23527 and U.S. Pat.No. 5,741,705. Examples of a yeast-based hydrolysate which also may beused in the invention include TC Yeastolate (BD Diagnostic) andYeastolate UF (SAFC Biosciences), while examples of plant-basedhydrolysates include Soy Hydrolysate UF (SAFC Biosciences) and HyQ® SoyHydrolysate (HyClone Media).

In one embodiment, the cell culture medium of the invention furtherincludes glutamine, e.g, L-glutamine. Suitable sources of L-glutamineare available from various commercial sources, such as Gibco (Cat. No.25030-081).

Optionally, the cell culture medium of the invention, including thosedescribed below in the Examples section, may include methotrexate.Examples of amounts of methotrexate used in the cell culture media forculturing CHO cells include about 100 nM to 5000 nM methotrexate.Numbers intermediate to the recited methotrexate molarity, e.g., 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800,2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200,4400, 4600, 4800, and 5000 nM, as well as numbers intermediate thereto,are also intended to be part of this invention. Ranges of values using acombination of any of the above recited values as upper and/or lowerlimits are also intended to be included in the scope of the invention.

In large scale bioreactors, CHO cells are particularly susceptible tosheer forces arising from the sparging of the vessel with gases and themixing with the impeller. Thus, cell culture media of the invention mayalso optionally include a cell protectant. The term “cell protectant” asused herein means a substance that protects eukaryotic cells fromdamage. Such damage may be caused, for example, by shear forces or theeffects of gas bubble sparging in a bioreactor vessel. To minimize theoccurrence of cellular damage it is advantageous for the medium tocontain a cell protectant, such as methyl cellulose, polyethyleneglycol, polyvinyl alcohols or pluronic polyols. Of these, Pluronic®(polyol, BASF Wyandotte Corp.) polyol F68 is preferred since unlikepolyvinyl alcohols this is a non-toxic substance and unlike polyethyleneglycols does not interfere with downstream purification.

The cell culture medium of the invention may also include non-ferrousmetal ions. Examples of non-ferrous metal ions include, but are notlimited to, chloride and sulfate salts, potassium, magnesium, cupric,selenium, zinc, nickel, manganese, tin, cadmium, molybdate, vanadate,and silicate.

The cell culture medium of the invention may also include vitamins andenzyme co-factors. Examples of such vitamins and enzyme co-factorsinclude, but are not limited to, PABA (p-Aminobenzoic Acid), Vitamin K(Biotin), Vitamin B5 (D-Calcium Pantothenate), Folic Acid, I-Inositol,Niacinamide (Niccotinic Acid Amide), Vitamin B6 (PyrdoxineHCl) (andPyrodoxal HCl), Vitamin B2 (Riboflavin), Vitamin B1 (Thiamine), andVitamin B12 (Cyanocobalamin). Alternatively, vitamin C (L-Ascorbic Acid)may be added to the media. Choline Chloride may also be added, it isusually considered a vitamin but it may also be considered a lipidfactor.

Additionally, the cell culture medium of the invention may also includelipid like factors. Examples of lipid factors include choline chlorideand phosphatidylcholine. An aid in lipid production, e.g., an alcoholamine like ethanolamine, may also be included.

In the methods and compositions of the invention, cells are preferablycultured in serum-free media. The term “serum-free” as applied to mediaincludes any mammalian cell culture medium that does not contain serum,such as fetal bovine serum.

Also included within the scope of the invention are mammalian cells,e.g., CHO cells, in any of the improved cell culture media describedherein.

In one aspect of the invention, formulations for cell culture mediaoptimized for the production of a certain antibody are provided.Examples of optimized formulations include cell culture media for thegrowth or protein production of CHO cells which express an anti-TNFαantibody, an anti-IL-12 antibody, an anti-IL-18 antibody, and ananti-EPO receptor (EPO-R) antibody.

Included in the invention are cell culture media for mammalian cells,e.g., CHO cells, which express anti-TNFα antibodies, including fullyhuman anti-TNFα antibodies. In a preferred embodiment, the fully humananti-TNFα antibody is adalimumab (also referred to as D2E7 and Humira®:Abbott Laboratories). Characteristics of adalimumab, including nucleicacid and amino acid sequences, are described in U.S. Pat. No. 6,090,382,incorporated by reference herein. Adalimumab may be produced byculturing mammalian cells, e.g., CHO cells, comprising a nucleic acidencoding the protein, e.g., adalimumab, in a cell culture growth medium,transferring the cell culture into a cell culture production medium, andisolating the protein from the cell culture production medium.

In one embodiment, the invention provides a cell culture growth mediumoptimized for CHO cells comprising a nucleic acid encoding adalimumab.The formulation of serum-free cell culture growth medium optimized forCHO cells expressing adalimumab includes a basal medium; ferric citrate(e.g., about 8-12 ml/kg, about 10.0 ml/kg, or 122 mg/L); recombinanthuman insulin (e.g., about 2-6 mg/kg, or 4.0 mg/kg); anhydrous glucose(e.g., 2-5 g/kg, about 3.5 g/kg); L-glutamine (e.g., about 0.29 g/kg);sodium bicarbonate (e.g., about 1.6 g/kg); NaH₂PO₄.H₂O (e.g., about 0.03g/kg); Na₂HPO₄.7H₂O (e.g., about 0.43 to 0.44 g/kg); and yeast-basedhydrolysate (e.g., about 2.0 g/kg). Another example of a serum-free cellculture growth medium optimized for CHO cells expressing adalimumabincludes the following ingredients: a modified basal medium, whichexcludes the following components sodium bicarbonate, buffer, mono-basicsodium phosphate, di-basic sodium phosphate, an osmolarity regulator, asurfactant, and monosaccharide glucose; ferric citrate (e.g., about 10.0ml/kg or 122.45 mg/kg); recombinant human insulin (e.g., about 3.8 to3.9 mL/kg or 7.8 mg/kg); anhydrous glucose (e.g., about 7.0 g/kg);L-glutamine (e.g., about 0.8 to 0.9 g/kg); sodium bicarbonate (e.g.,about 1.6 g/kg); HEPES (e.g., about 1.8 g/kg); NaCl (e.g., about 2.6 to2.7 g/kg); Pluronic F-68 (e.g., about 1.0 g/kg); NaH₂PO₄.H₂O (e.g.,about 0.03 to 0.04 g/kg); Na₂HPO₄.7H₂O (e.g., about 0.43 to 0.44 g/kg);L-asparagine monohydrate (e.g., about 0.45 g/kg); yeast-basedhydrolysate (e.g., about 4.0 g/kg); and plant-based hydrolysate (e.g.,about 2.6 g/kg). The cell culture growth medium for expressingadalimumab may further include methotrexate, e.g., about 1-5 mL/kg, orabout 2.50 mL/kg.

In one embodiment, the invention provides a cell culture productionmedium optimized for CHO cells expressing an antibody, including, forexample, a human anti-TNFα antibody (e.g., adalimumab) or anerythropoietin receptor (EPO/R) antibody. Examples of anti-EPO/Rantibodies are described in US Patent Publication No. 20040071694,hereby incorporated by reference. The formulation of serum-free cellculture production medium optimized for CHO cells expressing antibodies,such as adalimumab or an anti-EPO/R antibody, includes a modified basalmedium which excludes the following components sodium bicarbonate,buffer, mono-basic sodium phosphate, di-basic sodium phosphate, anosmolarity regulator, a surfactant, and monosaccharide glucose; ferriccitrate (e.g., about 10.0 ml/kg or 122.45 mg/L); recombinant humaninsulin (e.g., about 6.0 mL/kg or 12 mg/kg); anhydrous glucose (e.g.,about 7.0 g/kg); L-glutamine (e.g., about 0.58 to 0.59 g/kg); sodiumbicarbonate (e.g., about 1.6 g/kg); HEPES (e.g., about 1.8 g/kg); NaCl(e.g., about 2.4 to 2.5 g/kg); Pluronic F-68 (e.g., about 1.0 g/kg);NaH₂PO₄.H₂O (e.g., about 0.03 to 0.04 g/kg); Na₂HPO₄.7H₂O (e.g., about0.43 to 0.44 g/kg); a yeast-based hydrolysate (e.g., bout 10.7 g/kg);and plant-based hydrolysate (e.g., about 6.9 to 7.0 g/kg). In oneembodiment, the cell culture production medium has a pH ranging fromabout 7.1 to 7.2 and an osmolality ranging from 373 to 403 mOsm/kg.Numbers intermediate to the above values, e.g., recited insulin, pH, orosmolality values, are also intended to be part of this invention.

Another aspect of the invention relates to cell culture media which isoptimized the production anti-interleukin-12 (IL-12) antibodies, e.g.,fully human IL-12 antibodies, produced by CHO cells. In a preferredembodiment, the fully human anti-IL12 antibody is ABT-874.Characteristics of ABT-874, including the nucleic acid and amino acidsequences, are described in U.S. Pat. No. 6,914,128, incorporated byreference herein.

In one embodiment, a serum-free cell culture growth medium optimized forgrowth of CHO cells expressing ABT-874 includes the following: amodified basal medium, which excludes the following components sodiumbicarbonate, buffer, mono-basic sodium phosphate, di-basic sodiumphosphate, an osmolarity regulator, a surfactant, and monosaccharideglucose; ferric citrate (e.g., about 10 ml/kg or 122.45 mg/L);recombinant human insulin (e.g., about 3.8 to 3.9 mL/kg or 7.8 mg/kg);anhydrous glucose (e.g., about 7.0 g/kg); L-glutamine (e.g., about 0.87to 0.88 g/kg); L-asparagine monohydrate (e.g., about 0.45 g/kg); sodiumbicarbonate (e.g., about 1.6 g/kg); HEPES (e.g., about 1.8 g/kg); NaCl(e.g., about 2.67 to 2.68 g/kg); about Pluronic F-68 (e.g., 1.0 g/kg);NaH₂PO₄.H₂O (e.g., about 0.03 to 0.04 g/kg); Na₂HPO₄.7H₂O (e.g., about0.43 to 0.44 g/kg); yeast-based hydrolysate (e.g., about 4.0 g/kg); andplant-based hydrolysate (e.g., about 2.6 g/kg).

In one embodiment, a serum-free cell culture production medium forexpression of ABT-874 includes the following: a modified basal mediumhaving reduced vitamin content and excluding the following componentssodium bicarbonate, buffer, mono-basic sodium phosphate, di-basic sodiumphosphate, an osmolarity regulator, a surfactant, and monosaccharideglucose; ferric citrate (e.g., about 10 ml/kg or 122.45 mg/L);recombinant human insulin (e.g., about 6.5 mL/kg or 13 mg/kg); anhydrousglucose (e.g., about 7.0 g/kg); L-glutamine (e.g., about 0.58 to 0.59g/kg); sodium bicarbonate (e.g., about 1.6 g/kg); HEPES (e.g., about 1.8g/kg); NaCl (e.g., about 2.45 g/kg); Pluronic F-68 (e.g., about 1.0g/kg); NaH₂PO₄.H₂O (e.g., about 0.03 to 0.04 g/kg); Na₂HPO₄.7H₂O (e.g.,about 0.43 to 0.44 g/kg); yeast-based hydrolysate (e.g., about 10.7g/kg); and a plant-based hydrolysate (e.g., about 6.9 to 7.0 g/kg).

In one embodiment, the invention provides a serum free cell culturegrowth medium for CHO cells expressing antibodies, e.g., anti-IL12 andanti-EPO/R antibodies, comprising the following: a basal medium; ferriccitrate (e.g., about 10 ml/kg or 122.45 mg/L); recombinant human insulin(e.g., about 4 mg/kg); anhydrous glucose (e.g., about 1.5 g/kg);L-glutamine (e.g., about 0.29 to 0.30 g/kg); sodium bicarbonate ((e.g.,about 1.6 g/kg); and yeast-based hydrolysate (e.g., at least about 2g/kg). In one embodiment, the cell culture growth medium for CHO cellsexpressing antibodies, e.g., anti-IL12 and anti-EPO/R antibodies, has apH of about 7.10 to 7.30 and an osmolality of about 300 to 340 mOsmo/kg.The cell culture medium may also contain a yeast-based hydrolysate (e.g,at least about 8 g/kg).

Another aspect of the invention relates to cell culture media optimizedfor the production of anti-interleukin-18 (IL-18) antibodies, e.g.,fully human IL-18 antibodies, produced by CHO cells. Examples of fullyhuman IL-18 antibodies which may be produced using the methods andcompositions of the invention are described in PCT publication WO01/058956, incorporated by reference herein.

In one embodiment, a cell culture growth medium optimized for IL-18antibody production in CHO cells includes the following ingredients: amodified basal medium which excludes the following components sodiumbicarbonate, buffer, mono-basic sodium phosphate, di-basic sodiumphosphate, an osmolarity regulator, a surfactant, and monosaccharideglucose; ferric citrate (e.g., about 10 ml/kg or 122.45 mg/L);recombinant human insulin (e.g., about 3.8 to 3.9 mL/kg or 7.8 mg/kg);anhydrous glucose (e.g., about 7.0 g/kg); L-glutamine (e.g., about 0.87to 0.88 g/kg); L-asparagine monohydrate (e.g., about 0.45 g/kg); sodiumbicarbonate (e.g., about 1.6 g/kg); HEPES (e.g., about 1.8 g/kg); NaCl(e.g., about 2.67 g/kg); Pluronic F-68 (e.g., about 1.0 g/kg);NaH₂PO₄.H₂O (e.g., about 0.03 to 0.04 g/kg); Na₂HPO₄.7H₂O (e.g., about0.43 to 0.44 g/kg); yeast-based hydrolysate (e.g., about 4.0 g/kg); anda plant-based hydrolysate (e.g., about 2.6 g/kg). The cell culturemedium for growth of cells expressing fully human IL-18 antibodies mayhave a pH of 7.10 to 7.20 and an osmolality of 373 to 403 mOsm/kg.Numbers intermediate to the above values, e.g., recited insulin, pH, orosmolality values, are also intended to be part of this invention.

An example of a cell culture production medium optimized for IL-18antibody production in CHO cells includes the following ingredients: amodified basal medium, which is modified to remove the followingcomponents sodium bicarbonate, HEPES buffer, mono-basic sodiumphosphate, di-basic sodium phosphate, an osmolarity regulator, asurfactant, and monosaccharide glucose; ferric citrate (e.g., about 10ml/kg or 122.45 mg/L); recombinant human insulin (e.g., about 6.0 mL/kgor 12 mg/kg); anhydrous glucose (e.g., about 7.0 g/kg); L-glutamine(e.g., about 0.58 to 0.59 g/kg); sodium bicarbonate (e.g., about 1.6g/kg); HEPES (e.g., about 1.8 g/kg); NaCl (e.g., about 2.45 g/kg);Pluronic F-68 (e.g., about 1.0 g/kg); NaH₂PO₄.H₂O (e.g., about 0.03 to0.04 g/kg); Na₂HPO₄.7H₂O (e.g., about 0.43 to 0.44 g/kg); yeast-basedhydrolysate (e.g., about 10.7 g/kg); and a plant-based hydrolysate(e.g., about 6.9 to 7.0 g/kg). Another example of a cell cultureproduction medium for producing fully human anti-IL-18 antibodiesincludes a modified basal medium which excludes the following componentssodium bicarbonate, buffer, mono-basic sodium phosphate, di-basic sodiumphosphate, an osmolarity regulator, a surfactant, and monosaccharideglucose; ferric citrate (e.g., about 10 ml/kg or 122.45 mg/L);recombinant human insulin (e.g., a about 6.5 mL/kg or 13 mg/kg);anhydrous glucose (e.g., about 7.0 g/kg); L-glutamine (e.g., about 0.58to 0.59 g/kg); sodium bicarbonate (e.g., about 1.6 g/kg); HEPES (e.g.,about 1.8 g/kg); NaCl (e.g., about 2.45 g/kg); Pluronic F-68 (e.g.,about 1.0 g/kg); NaH₂PO₄.H₂O (e.g., about 0.03 to 0.04 g/kg);Na₂HPO₄.7H₂O (e.g., about 0.43 to 0.44 g/kg); yeast-based hydrolysate(e.g., about 14.2 to 14.3 g/kg); and a plant-based hydrolysate (e.g.,about 9.2 to 9.3 g/kg). The cell culture medium for CHO cells producingfully human IL-18 antibodies may have a pH of 7.10 to 7.20 and anosmolality of 373 to 403 mOsm/kg. Numbers intermediate to the abovevalues, e.g., recited insulin, pH, or osmolality values, are alsointended to be part of this invention.

Other examples of media within the scope of the invention are describedbelow relating to fed batch methods and supplemental media which improveantibody production, in addition to the exemplary media described in theExamples section.

The media of the present invention may be used to effect appropriateculture of cells by bringing the media or its components into contactwith all or part of the cell population. The media may be brought intocontact with the cells by mixing, adding, combining, seeding, orstirring of one or more cells with one or more compounds, solutions,media, etc. Media may also be brought into contact with the cells all atonce, incrementally, or in a step-wise manner by, for example, “feeding”to replace or supplement the medium in which cells are cultured,described in more detail below.

IV. Methods and Compositions for Improved Protein Production

The methods of compositions of the invention are directed towardmammalian cell culture. In one embodiment, the mammalian cell used isthe CHO cell.

Established methods for introducing DNA into mammalian cells have beendescribed. Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp.15-69. Additional protocols using commercially available reagents, suchas the cationic lipid reagents Lipofectamine™ Lipofectamine™-2000, orLipofectamine™-plus (which can be purchased from Invitrogen), can beused to transfect cells. Feigner et al. (1987), Proc. Natl. Acad. Sci.USA 84:7413-7417. In addition, electroporation or bombardment withmicroprojectiles coated with nucleic acids can be used to transfectmammalian cells using procedures, such as those in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed. Vol. 1-3, Cold SpringHarbor Laboratory Press (1989) and Fitzpatrick-McElligott (1992),Biotechnology (NY) 10(9): 1036-40. Selection of stable transformants canbe performed using methods known in the art, such as, for example,resistance to cytotoxic drugs. Kaufman et al. ((1990), Meth. inEnzymology 185:487-511), describes several selection schemes, such asdihydrofolate reductase (DHFR) resistance. A suitable host strain forDHFR selection can be CHO strain DX-B11, which is deficient in DHFR.Urlaub and Chasin (1980), Proc. Natl. Acad. Sci. USA 77:4216-4220. Aplasmid expressing the DHFR cDNA can be introduced into strain DX-B11,and only cells that contain the plasmid can grow in the appropriateselective media. Other examples of selectable markers that can beincorporated into an expression vector include cDNAs conferringresistance to antibiotics, such as G418 and hygromycin B. Cellsharboring the vector can be selected on the basis of resistance to thesecompounds.

Additional control sequences shown to improve expression of heterologousgenes from mammalian expression vectors include such elements as theexpression augmenting sequence element (EASE) derived from CHO cells(Morris et al., in Animal Cell Technology, pp. 529-534 (1997); U.S. Pat.Nos. 6,312,951 B1, 6,027,915, and 6,309,841 B1) and the tripartiteleader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras et al. (1982),J. Biol. Chem. 257:13475-13491). The internal ribosome entry site (IRES)sequences of viral origin allows dicistronic mRNAs to be translatedefficiently (Oh and Sarnow (1993), Current Opinion in Genetics andDevelopment 3:295-300; Ramesh et al. (1996), Nucleic Acids Research24:2697-2700). Expression of a heterologous cDNA as part of adicistronic mRNA followed by the gene for a selectable marker (e.g.DHFR) has been shown to improve transfectability of the host andexpression of the heterologous cDNA (Kaufman et al. (1990), Methods inEnzymol. 185:487-511). Exemplary expression vectors that employdicistronic mRNAs are pTR-DC/GFP described by Mosser et al.,Biotechniques 22:150-161 (1997), and p2A5I described by Morris et al.,in Animal Cell Technology, pp. 529-534 (1997).

A useful high expression vector, pCAVNOT, has been described by Mosleyet al. ((1989), Cell 59:335-348). Other expression vectors for use inmammalian host cells can be constructed as disclosed by Okayama and Berg((1983), Mol. Cell. Biol. 3:280). A useful system for stable high levelexpression of mammalian cDNAs in C127 murine mammary epithelial cellscan be constructed substantially as described by Cosman et al. ((1986),Mol. Immunol. 23:935). A useful high expression vector, PMLSV N1/N4,described by Cosman et al. ((1984), Nature 312:768), has been depositedas ATCC 39890. Additional useful mammalian expression vectors aredescribed in EP Patent No.-A-0 367 566 and WO 01/27299 A1. The vectorscan be derived from retroviruses. In place of the native signalsequence, a heterologous signal sequence can be added, such as one ofthe following sequences: the signal sequence for IL-7 described in U.S.Pat. No. 4,965,195; the signal sequence for IL-2 receptor described inCosman et al. (Nature 312:768 (1984)); the IL-4 signal peptide describedin EP Patent No. 0 367 566; the typed IL-1 receptor signal peptidedescribed in U.S. Pat. No. 4,968,607; and the type II IL-1 receptorsignal peptide described in EP Patent No. 0 460 846.

The polypeptides can be produced recombinantly in eukaryotic cells, andare preferably secreted by host cells adapted to grow in cell culture.Host cells for use in the invention are preferably mammalian cells. Thecells can be also genetically engineered to express a gene of interest,can be mammalian production cells adapted to grow in cell culture,and/or can be homogenous cell lines. Examples of such cells commonlyused in the industry are VERO, BHK, HeLa, CV1 (including Cos), MDCK,293, 3T3, myeloma cell lines (e.g., NSO, NS1), PC12, WI38 cells, andChinese hamster ovary (CHO) cells, which are widely used for theproduction of several complex recombinant polypeptides, e.g. cytokines,clotting factors, and antibodies (Brasel et al. (1996), Blood88:2004-2012; Kaufman et al. (1988), J. Biol. Chem. 263:6352-6362;McKinnon et al. (1991), J Mol Endocrinol 6:231-239; Wood et al. (1990),J. Immunol. 145:3011-3016). The dihydrofolate reductase (DHFR)-deficientmutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77:4216-4220, which is incorporated by reference), DXB11 and DG-44, aredesirable CHO host cell lines because the efficient DHFR selectable andamplifiable gene expression system allows high level recombinantpolypeptide expression in these cells (Kaufman R. J. (1990), MethEnzymol 185:537-566, which is incorporated by reference). In addition,these cells are easy to manipulate as adherent or suspension culturesand exhibit relatively good genetic stability. CHO cells and recombinantpolypeptides expressed in them have been extensively characterized andhave been approved for use in clinical commercial manufacturing byregulatory agencies. The methods of the invention can also be practicedusing hybridoma cell lines that produce an antibody. Methods for makinghybridoma lines are well known in the art. See e.g. Berzofsky et al. inPaul, ed., Fundamental Immunology, Second Edition, pp. 315-356, at347-350, Raven Press Ltd., New York (1989). Cell lines derived from theabove-mentioned lines are also suitable for practicing the invention.

Following transformation of a suitable mammalian host cell, e.g., CHOcell, with polynucleotide sequences encoding a recombinant protein,cells demonstrating stable expression of the recombinant protein areidentified and isolated. Stable expression of a recombinant protein isachieved by transfection of appropriate DNA vectors into dihydrofolatereductase deficient (DHFR−) Chinese hamster ovary cells (CHO AM-1/D,U.S. Pat. No. 6,210,924) followed by isolation and testing of individualclones demonstrating highest expression of recombinant protein, inaccordance with methods known in the art. Based on growth and productionin small-scale spinners and larger scale bioreactors, a specific cellline is chosen as the cell line for manufacturing of the recombinantprotein.

Cells producing the highest levels of recombinant protein may be clonedby methods well-known in the art, for example, by multiple rounds oflimiting dilution in 96 and/or 24 well plates under serum-freeconditions, using the cell culture media of the present invention. Theclones are selected based on production and growth characteristics invarious suspension vessels. Enzyme Immunoassays (EIAs) may be performedto select the clone that produces the highest level of recombinantprotein. Growth characteristics, including doubling times and densitiesmay be measured by growing the clones various shaker or spinner flasksand bioreactors ranging from 100 ml to up to 3 L. An optimal clone, forexample a clone with the fastest doubling time that reaches the highestdensity in culture, is selected, and is selected as the cell line foruse in recombinant protein production. In one embodiment, therecombinant protein production is for commercial purposes and isperformed using a large-scale bioreactor.

Typically, cell culture is performed under sterile, controlledtemperature and atmospheric conditions in tissue culture plates (e.g.,10-cm plates, 96-well plates, etc.), or other adherent culture (e.g., onmicrocarrier beads) or in suspension culture such as in roller bottles.Cultures can be grown in shake flasks, small scale bioreactors, and/orlarge-scale bioreactors. A bioreactor is a device used to culture cellsin which environmental conditions such as temperature, atmosphere,agitation, and/or pH can be monitored, adjusted and controlled. Themethods, including fed batch methods, and compositions of the inventionmay be used in large scale mammalian cell culture, e.g., 10 L, 11 L, 12L, 13 L, 14 L, and so forth. In one embodiment, the large scale cellculture methods and compositions of the invention are suitable for CHOcell culture and antibody production.

According to the present invention, a mammalian host cell is culturedunder conditions that promote the production of the polypeptide ofinterest, which can be an antibody or a recombinant polypeptide. Theskilled artisan may choose to use one or more of cell culture mediadescribed herein that have been developed to maximize cell growth, cellviability, and/or recombinant polypeptide production in a particularcultured host cell. Alternatively, the methods and compositionsaccording to the current invention may be used in combination withcommercially available cell culture media.

Suitable culture conditions for mammalian cells are known in the art(see e.g. Animal cell culture: A Practical Approach, D. Rickwood, ed.,Oxford university press, New York (1992)), and may be combined with theimproved methods of the invention. Mammalian cells may be cultured insuspension or while attached to a solid substrate. Furthermore,mammalian cells may be cultured, for example, in fluidized bedbioreactors, hollow fiber bioreactors, roller bottles, shake flasks, orstirred tank bioreactors, with or without microcarriers, and operated ina batch, fed batch, continuous, semi-continuous, or perfusion mode.

The methods according to the present invention may be used to improvethe production of recombinant polypeptides in both single phase andmultiple phase culture processes. In a single phase process, cells areinoculated into a culture environment and the disclosed methods areemployed during the single production phase. In a multiple stageprocess, cells are cultured in two or more distinct phases. For examplecells may be cultured first in a growth phase, under environmentalconditions that maximize cell proliferation and viability, thentransferred to a production phase, under conditions that maximizepolypeptide production. The growth and production phases may be precededby, or separated by, one or more transition phases. In multiple phaseprocesses the methods according to the present invention are employed atleast during the production phase.

For the purposes of understanding, yet without limitation, it will beappreciated by the skilled practitioner that cell cultures and culturingruns for protein production can include three general types; namely,continuous culture, batch culture and fed-batch culture. In a continuousculture, for example, fresh culture medium supplement (i.e., feedingmedium) is provided to the cells during the culturing period, while oldculture medium is removed daily and the product is harvested, forexample, daily or continuously. In continuous culture, feeding mediumcan be added daily and can be added continuously, i.e., as a drip orinfusion. For continuous culturing, the cells can remain in culture aslong as is desired, so long as the cells remain alive and theenvironmental and culturing conditions are maintained.

In batch culture, cells are initially cultured in medium and this mediumis neither removed, replaced, nor supplemented, i.e., the cells are not“fed” with new medium, during or before the end of the culturing run.The desired product is harvested at the end of the culturing run.

For fed-batch cultures, the culturing run time is increased bysupplementing the culture medium one or more times daily (orcontinuously) with nutrient solutions during the run, i.e., the cellsare “fed’ with feeding medium during the culturing period. Fed-batchcultures can include the various feeding regimens and times as describedbelow, for example, daily, every other day, every two days, etc., morethan once per day, or less than once per day, and so on. Further,fed-batch cultures can be fed continuously with feeding medium. Thedesired product is then harvested at the end of the culturing/productionrun. The present invention preferably embraces fed-batch cell cultures,with feeding using optimized feeding solutions which increase proteinproduction and can extend the protein production phase.

Improved Fed Batch Culture: Hydrolysate and Basal Enrichment Solutions

One aspect of the invention features methods and compositions forincreasing protein production using an improved fed batch method incombination with supplemental basal and hydrolysate solutions. Theimproved fed batch method, in part, is based on the addition of twoenrichment solutions, i.e., a hydrolysate enrichment solution and abasal enrichment solution, which are added to the cell culture mediumduring a time period during protein production. The hydrolysateenrichment solution used in the fed batch method of the inventioncomprises a first hydrolysate which is not derived from a plant or ananimal and a second plant-based hydrolysate. An example of a hydrolysatewhich is not derived from a plant or an animal and a plant-basedhydrolysate is a yeast-based hydrolysate. An example of a plant-basedhydrolysate which may be used in the hydrolysate enrichment solution isa soy-based hydrolysate. The basal enrichment solution includes a basalmedium, e.g., PF CHO, asparagine, and glucose, and the hydrolysateenrichment solution includes at least two different non-animal-basedhydrolysates. The hydrolysate and basal enrichment solutions are addedto the cell culture at intervals during a time period, e.g., dailyintervals during an 11-15 day time period, and may be added on the sameday or on different days.

The invention features a fed batch method for producing an anti-TNFαantibody, e.g., adalimumab/D2E7, comprising culturing Chinese HamsterOvary (CHO) cells comprising a nucleic acid encoding the anti-TNFαantibody in a cell culture at a culturing temperature of ranging from 32to 38° C. In one embodiment, the culturing temperature is 35° C. The CHOcells are fed a hydrolysate enrichment solution and a basal enrichmentsolution in order to address, e.g., solve or correct, nutritionaldeficiencies to maximize productivity. The hydrolysate and basalenrichment solutions are added to the cell culture. In one embodiment,the cell culture production medium comprising between 20 and 65%dissolved oxygen, e.g., about 30% dissolved oxygen. In one embodiment,the cell culture production medium contain a level of glucose needed forprotein production, e.g., at least 1-5 g/L of glucose. In oneembodiment, the cell culture production medium contains about 1.5-2.5g/L of glucose. In a further embodiment, the cell culture productionmedium contains about 2.0 g/L of glucose. The glucose concentration maybe controlled throughout the protein production culturing process byadding glucose to the cell culture production medium as required tomaintain a given concentration, e.g., at least 2.0 g/L of glucose. Inone embodiment, the hydrolysate enrichment solution used in the fedbatch method for an anti-TNFα antibody comprises of about 50-280 g/L ofa soy-based hydrolysate (including ranges and numbers therein, e.g.,100-225, 50-225, 255-275, and 265 g/L), and about 75-300 g/L of ayeast-based hydrolysate (including ranges and numbers therein, e.g.,100-250, 150-200, 145-175, and 165 g/L).

Basal enrichment solution optimized for use in a fed batch method forthe production of an anti-TNFα antibody, e.g, adalimumab/D2E7, in CHOcells has a pH of about 9.0 to 10.5 (including ranges and numberstherein, e.g., 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1,10.2, 10.3, 10.4, 10.5). The time period during which the hydrolysateenrichment solution and the basal enrichment solution are added isbetween 9 to 15 days, e.g., 12 days, In one embodiment, the basalenrichment solution is added to the cell culture medium on at least oneof the following days of the time period: Day 4, Day 6, Day 9, and Day11, and the hydrolysate enrichment solution is added to the cell culturemedium on Day 4, Day 7, or Day 4 and Day 7 of the time period. Numbersintermediate to the above values are also intended to be part of thisinvention.

In another alternative, the fed batch method for the production of ananti-TNFα antibody, e.g., adalimumab/D2E7, may include adjusting the pHof the cell culture medium according to a pH linear ramp comprisingstarting from a pH of about 6.5-8, e.g., 7.1 to 7.2, and resulting in afinal pH of about 6.9. In one embodiment, the pH linear ramp is adjustedover a period of at least about 24 hours, 48 hours, or 72 hours.

The invention also features a fed batch method of producing an anti-IL12antibody, e.g., ABT-874, comprising culturing Chinese Hamster Ovary(CHO) cells comprising a nucleic acid encoding the anti-IL12 antibody ina cell culture at a culturing temperature of ranging from 32 to 38° C.,e.g., 33° C. The hydrosylate enrichment solution used for IL-12 antibodyproduction may also contain glucose. In one embodiment, the CHO cellsare cultured at a pH of about 6.9. The CHO cells are fed a hydrolysateenrichment solution and a basal enrichment solution in order to maintainnutritional deficiencies to maximize productivity. The hydrolysate andbasal enrichment solutions are added to the cell culture. In oneembodiment, the cell culture production medium comprising between 20 and65% dissolved oxygen, e.g., about 40% dissolved oxygen. In oneembodiment, the hydrolysate enrichment solution used in the fed batchmethod for an anti-IL12 antibody comprises about 50-280 g/L of asoy-based hydrolysate (including ranges and numbers therein, e.g.,100-225, 50-225, 255-275, and 265 g/L), about 75-300 g/L of ayeast-based hydrolysate (including ranges and numbers therein, e.g.,100-250, 150-200, 145-175, and 165 g/L), and about 2-3 g/L of glucose,e.g., 2.4 g/L glucose. Basal enrichment solution optimized for use in afed batch method for the expression of an anti-IL12 antibody, e.g.,ABT-874, in CHO cells has a pH of about 9.7 and an osmolarity of about1480 mOsm. The time period during which the hydrolysate enrichmentsolution and the basal enrichment solution are added is between 14 to 15days, e.g., 12 days. In one embodiment, the basal enrichment solution isadded to the cell culture production medium every other day beginning onday 5 of the time period, and the hydrolysate enrichment solution isadded to the cell culture production medium every day beginning on day 6of the time period. Alternatively, the basal enrichment solution and thehydrolysate enrichment solution may be added to the cell cultureproduction medium every day beginning on day 5 of the time period.Numbers intermediate to the above range values are also intended to bepart of this invention.

Stable, High Concentration Feed Solution

One aspect of the invention features methods and compositions relatingto an improved, stable high concentration feed solution for improvingprotein productivity. The improved feed solution may be used tosupplement cell culture production medium in the production of anantibody. The feed solution includes glucose (e.g., 100 to 250 g/kg); abasal medium; an amino acid other than glutamine, e.g., asparagine(e.g., 1.0 to 15.0 g/kg; 3-12.5 g/kg, or 3-5 g/kg); and at least twodifferent non-animal based hydroslyates. In addition, the feed solutionhas a pH of about 6.0 to 7.5. The two different non-animal basedhydrolysates may include a plant-based hydrolysate, e.g, soy-basedhydrolysate, and a hydrolysate which is not animal-based or plant based,e.g., a yeast-based hydrolysate. Any basal medium known in the art maybe used in the improved feed solution, including, but not limited to, PFCHO or DMEM/F12 medium. A modified basal medium may also be used. In oneembodiment, the basal cell medium excludes the following components:sodium bicarbonate, buffer, mono-basic sodium phosphate, di-basic sodiumphosphate, an osmolarity regulator, a surfactant, glutamine, andglucose. The improved feed solution is stable, having a turbidity ofless than about 15 NTU. The invention also includes maintaining a steadyglucose level of a cell culture production medium by adding the feedsolution. Numbers intermediate to the above range values are alsointended to be part of this invention, e.g., 3.0, 3.1, 3.2, 3.3, 3.6,3.8, 4, 4.2, 4.4, 4.6, 4.8, and 5 g/kg asparagine.

Also included in the invention is a method for making a feed solutioncomprising glucose and at least two different non-animal basedhydrolysates. The method for making the feed solution includes combiningglucose and a basal cell medium into a combination solution, andadjusting the pH of the combination solution to about 9.5 to 10.5. Atleast two different non-animal based hydrolysates are then added to thesolution, and the pH is adjusted again such that the resulting feedsolution has a pH of about 6.5 to 7.5. Numbers intermediate to the aboverange values are also intended to be part of this invention, e.g., pH of6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5.

The feed solution of the invention may be used to achieve high levels ofrecombinant protein production, e.g., antibody production, frommammalian cell culture. In one embodiment, the invention features amethod for producing at least 1.5 g/L of an antibody from a mammaliancell culture comprising culturing mammalian cells comprising a nucleicacid encoding the antibody in a cell culture production medium. A feedsolution having a pH of about 6.7 to 7.0 is then added to the cellculture production medium. The feed solution includes glucose (e.g.,about 100 to 250 g/kg); a basal cell medium; an amino acid other thanglutamine; and at least two different non-animal based hydrolysates. Inone embodiment, such a process results in at least 2 g/L of an antibodybeing produced; at least 4 g/L of an antibody being produced; at leastabout 5 g/L of the antibody; and at least 6 g/L of the antibody. Numbersintermediate to the above range values are also intended to be part ofthis invention, e.g., 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,2.5 g/L of antibody.

By using a feed solution having a pH of about 6.7 to 7.2 and includingthe following components: glucose; a basal cell medium; about an aminoacid other than glutamine; and at least two different non-animal basedhydrolysates, the titer of antibody produced from a mammalian cellculture can be increased. In one embodiment, adding the feed solution toa cell culture production medium for mammalian cells comprising anucleic acid encoding the antibody, results in an increase of at least50% more than a control mammalian cell culture which is cultured withoutaddition of the feed solution. In one embodiment, addition of the feedsolution results in a titer increase of at least 100% more than thecontrol. In one embodiment, addition of the feed solution results in atiter increase of at least 150% more than the control. The supplementalfeed solution may be added to the cell culture at a certain celldensity, such as when the cell density reaches at least 2.0×10⁶ cells/mLor when the cell density reaches at least 3.5×10⁶ cells/ml. Numbersintermediate to the above range values are also intended to be part ofthis invention. Ranges of values using a combination of any of the aboverecited values as upper and/or lower limits are intended to be includedin the scope of the invention.

As the fed batch methods of the invention serve to address, e.g., solveor correct, certain nutritional requirements of a cell culture for theproduction of a protein, e.g., an antibody, it may be advantageous tomonitor certain ingredients. Ingredients that may be monitored includeany metabolic indicator, i.e., an indicator of cell metabolism. In oneembodiment, the fed batch method of the invention comprises monitoringthe glucose level in the cell culture medium, including, monitoring theglucose level so that it is maintained at between 0.25-20 g/L. In oneembodiment, the glucose level is maintained at 0.5 to 5.5 g/L or 40.0-5.5 g/L. By monitoring glucose levels, cell metabolism can beindirectly monitored. As described in Example 3 below, glucose may beused as a metabolic indicator. In one embodiment, a cell culture may besupplemented with a nutritional component(s), e.g., hydrolysate, basedon the level of glucose. Methods for monitoring glucose levels are knownin the art and may include monitoring using an automated samplingdevice. Another example of a metabolic indicator that may be used isglutamine. Numbers intermediate to the above ranges, as well as allother numbers recited herein, are also intended to be part of thisinvention. Ranges of values using a combination of any of the aboverecited values as upper and/or lower limits are intended to be includedin the scope of the invention.

A feedback control system may be used to monitor a metabolic indicatorlevel in the cell culture production medium. In one embodiment, in orderto meet a desired parameter set-point, e.g., a pre-determined glucoselevel, a combination feed solution is added to the cell cultureproduction medium as determined by the feedback control system.

The feedback control system may also be used to determine a feed profilefor a given mammalian cell culture and the production of a protein ofinterest. In one embodiment, a method of determining a feed profile forproducing a protein in a mammalian cell culture comprises culturingmammalian cells comprising a nucleic acid encoding the protein andadding a feed solution, e.g., a combination feed solution, to the cellculture medium using a feedback control system to monitor a metabolicindicator. The feed solution is added to the cell culture productionmedium to meet a target metabolic indicator setpoint, e.g., a glucoselevel. Following the conclusion of the cell culture, the amount of thefeed solution added to the cell culture production medium per day isdetermined and provides a feed profile of when a feed solution should beadded to the cell culture. Once a feed profile is established,monitoring of the metabolic indicator is no longer needed for a givenmammalian cell culture producing a protein of interest. A feed profilepresents many advantages, including a decreased risk of contaminationsince frequent sampling is no longer required.

N-Acetylcysteine and Sodium Butyrate Methods and Compositions

As described above, the cell culture processes of this inventionadvantageously achieve an increased antibody titer. Another aspect ofthe invention for achieving protein productivity in mammalian cellculture is through the addition of sodium butyrate, N-acetylcysteine, ora combination thereof, to the cell culture medium. In one embodiment,the invention features a method of producing an antibody by addingsodium butyrate (e.g., 0.1 mM to 10 mM), N-acetylcysteine (e.g., 1 mM to80 mM), or a combination thereof, to the cell culture medium. In oneembodiment, the antibody titer is at least about 100 mg/L; at leastabout 150 mg/L; at least about 200 mg/L; at least about 250 mg/L; atleast about 300 mg/L; at least about 350 mg/L; or at least about 400mg/L.

The invention also features a method of producing an antibody in amammalian cell culture such that the titer of the antibody is improvedat least 10% over a control mammalian cell culture by adding sodiumbutyrate (e.g., final concentration of 0.1 mM to 10 mM),N-acetylcysteine (e.g., final concentration of 1 mM to 80 mM), or acombination thereof, to cell culture medium (control accordingly lackssodium butyrate, N-acetylcysteine, or the combination thereof). In oneembodiment, the antibody titer of the mammalian cell culture is improvedat least 29% over the control mammalian cell culture; at least 40% overthe control mammalian cell culture; at least 70% over the controlmammalian cell culture; or at least 90% greater than the controlmammalian cell culture. The sodium butyrate, N-acetylcysteine, or thecombination thereof may be added to the mammalian cell culture duringthe growth phase of the mammalian cell culture. In one embodiment, thesodium butyrate, N-acetylcysteine, or the combination thereof, is addedto the mammalian cell culture between days 4 and 7 of the culture time.In one embodiment, N-acetylcysteine is added, either alone or incombination with sodium butyrate, in an amount ranging from a finalconcentration of 5 mM to 80 mM, e.g., 20-60 mM, or about 10 mM.

The invention also features a method for extending longevity of amammalian cell culture by at least about 45% in comparison to a controlmammalian cell culture by adding about 0.1 mM to 10 mM ofN-acetylcysteine to the cell medium (control lacks this addition). Inone embodiment, the longevity of the mammalian cell culture is extendedat least about 35% in comparison to the control mammalian cell culture;or at least about 55% in comparison to the control mammalian cellculture.

It should be noted that cell culture media and improved culturingmethods described herein may be used separately or in combination withone another.

After culturing using the methods and compositions of the invention, theresulting expressed protein can then be recovered or collected. Inaddition the protein can purified, or partially purified, from suchculture or component (e.g., from culture medium or cell extracts orbodily fluid) using known processes. Fractionation procedures caninclude but are not limited to one or more steps of filtration,centrifugation, precipitation, phase separation, affinity purification,gel filtration, ion exchange chromatography, hydrophobic interactionchromatography (HIC; using such resins as phenyl ether, butyl ether, orpropyl ether), HPLC, or some combination of above.

For example, the purification of the polypeptide can include an affinitycolumn containing agents which will bind to the polypeptide; one or morecolumn steps over such affinity resins as concanavalin A-agarose,HEPARIN-TOYOPEARL (chromatography medium) or Cibacrom blue 3GA SEPHAROSE(agarose beads); one or more steps involving elution; and/orimmunoaffinity chromatography. The polypeptide can be expressed in aform that facilitates purification. For example, it may be expressed asa fusion polypeptide, such as those of maltose binding polypeptide(MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits forexpression and purification of such fusion polypeptides are commerciallyavailable from New England BioLab (Beverly, Mass.), Pharmacia(Piscataway, N.J.) and InVitrogen, respectively. The polypeptide can betagged with an epitope and subsequently purified by using a specificantibody directed to such epitope. One such epitope FLAG (epitope tag)is commercially available from Kodak (New Haven, Conn.). It is alsopossible to utilize an affinity column comprising a polypeptide-bindingpolypeptide, such as a monoclonal antibody to the recombinant protein,to affinity-purify expressed polypeptides. Other types of affinitypurification steps can be a Protein A or a Protein G column, whichaffinity agents bind to proteins that contain Fc domains. Polypeptidescan be removed from an affinity column using conventional techniques,e.g., in a high salt elution buffer and then dialyzed into a lower saltbuffer for use or by changing pH or other components depending on theaffinity matrix utilized, or can be competitively removed using thenaturally occurring substrate of the affinity moiety. In one embodiment,the antibodies produced using the methods and compositions of theinvention are purified in accordance with the methods described in U.S.application Ser. No. 11/732,918, incorporated by reference herein.

The desired degree of final purity depends on the intended use of thepolypeptide. The methods and compositions of the invention are suitablefor therapeutic uses of the protein of interest. Thus, a relatively highdegree of purity is desired when the polypeptide is to be administeredin vivo. In such a case, the polypeptides are purified such that nopolypeptide bands corresponding to other polypeptides are detectableupon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Itwill be recognized by one skilled in the pertinent field that multiplebands corresponding to the polypeptide can be visualized by SDS-PAGE,due to differential glycosylation, differential post-translationalprocessing, and the like. Most preferably, the polypeptide of theinvention is purified to substantial homogeneity, as indicated by asingle polypeptide band upon analysis by SDS-PAGE. The polypeptide bandcan be visualized by silver staining, Coomassie blue staining, or (ifthe polypeptide is radiolabeled) by autoradiography.

The invention also optionally encompasses further formulating theproteins. By the term “formulating” is meant that the proteins can bebuffer exchanged, sterilized, bulk-packaged and/or packaged for a finaluser. Such compositions can comprise an effective amount of the protein,in combination with other components such as a physiologicallyacceptable diluent, carrier, or excipient. The term “physiologicallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).

It should be noted that, with respect to the numbers recited herein,numbers that are intermediate to the above ranges, as well as all othernumbers recited herein, are also intended to be part of this invention.Ranges of values using a combination of any of the above recited valuesas upper and/or lower limits are intended to be included in the scope ofthe invention.

EXAMPLES

The following examples exemplify improved methods and compositions forculturing mammalian cells, including improved methods and compositionsfor expressing biologics in mammalian cells. An improved biochemicallydefined medium for culturing Chinese Hamster Ovary (CHO) cells used toexpress various recombinant biologics is disclosed below. Further, mediafor large-scale culture of cells in bioreactors run under variousconfigurations and scales, containing the same components but withdiffering component balance and enriched to allow for greater cellgrowth and increased product expression, are also exemplified.

Example 1 Improved Media for Culturing Mammalian Cells

Typically mammalian, e.g., Chinese Hamster Ovary (CHO), cell culturemedia is based on commercially available media formulations, such asDMEM, Ham's F12, or combinations of these media types. For theproduction of proteins in mammalian cells, a cell culture medium must besufficiently enriched to support increases in both cell growth andbiologics product expression. The following example describes animproved biochemically defined medium for culturing mammalian cells,i.e., Chinese Hamster Ovary (CHO) cells to express various recombinantbiologics, including antibodies.

Chinese Hamster Ovary (CHO) cells, deficient in dihydrofolate reductase[dhfr(−)] were adapted to grow in suspension in the absence of serum orany other material derived from an animal source. Cells were grown inthe absence of hypoxanthine and thymidine, in defined cell culturemedium obtained from a commercial source, JRH PF-CHO (Catalog #67147).Although the cell line was not deficient in glutamine synthase,additional glutamine was added to the culture medium.

CHO cell lines expressing biologics, such as antibodies to a giventarget, were generated using molecular biological techniques well knownin the art. Briefly, an expression vector capable of expressing theantibody of interest, and capable of expressing the dhfr enzyme genewere introduced into CHO cells using methods well known in the art.Transfected cells of interest were obtained by selecting the cells inthe presence of hypoxanthine and thymidine. Selected transformants werefurther cultured in increasing concentrations of methotrexate, toamplify the transfected genes and increase yield of expressed proteins.The improved cell culture medium used to culture the CHO cells isdescribed below.

Example 1.1 Medium for Chinese Hamster Ovary (CHO) Cell Culture

Generally, media formulations for culturing CHO cells were made up ofthree parts designated Parts A, B, and C. Part A was a basal medium andcomprised water, amino acids, vitamins, inorganic metal salts, traceelements, ethanolamine, putrescine, a surfactant, sodium pyruvate,glutathione, and 2-Mercaptoethanol. Part B comprised an inorganic ironsource; and Part C comprised recombinant growth factors, buffers, anosmolality regulator, an energy source, various non-ferrous metal ions,a surfactant, and hydrolysates. The medium components were mostlyinorganic or from a recombinant source and were highly purified. Themedia did not contain proteins, lipids and carbohydrates from animalsources. Complex hydrolysates were obtained from highly processed yeastand plant sources.

Part A of the medium included protein-free CHO (PF CHO) medium (JRH—SAFCBiosciences; JRH Cat #67147—also referred to as Original Part A). Thus,Part A included a basal medium, including water, amino acids andvitamins. The basal medium (PF CHO) was selected for modification andreformulation to support further increases in cell growth and expressedprotein productivity. PF CHO was modified to remove certain components,including sodium bicarbonate, HEPES buffer, mono-basic sodium phosphate,di-basic sodium phosphate, an osmolarity regulator, a surfactant, andmonosaccharide glucose (modified PF CHO is referred to herein asmodified Part A (also referred to as JRH Catalog #67411)). Thesemodifications were also made to facilitate improvements in cell cultureprocess conditions in large-scale bioreactors.

The Part B component (JRH) consisted of a concentrated ferric citratesolution that was added separately. The concentration of the Part Bcomponent was held constant in all CHO cell culture projects.

Part C components included a growth factor, e.g., insulin, the aminoacid glutamine, TC yeastolate and soy hydrolysate phytone, methotrexatenecessary to retain selective pressure, and the base NaOH and acid HClfor adjusting the pH after the media was hydrated during preparation.

The improved cell culture media formulations were made as follows.First, CHO cells were originally grown in PF-CHO media (SAFC-JRH CatalogNo. 67147) obtained from JRH. PF-CHO media (Catalog #67147) was furthermodified for use with the CHO cell lines as described below. Thismodified medium was designated by JRH with a new Catalog #67411. Thegoal of the modifications was to allow increasing concentrations of PartA of the cell culture medium, such that osmolarity and pH wereunaffected. The original Part A (Cat #67147) was also declared by themanufacturer (JRH) to be without Sodium Bicarbonate, Glutamine, andProtein-free (no Insulin or other protein or peptide growth factorsadded). These omitted components were then added to both 67147 and 67411Part A formulations independently, specifically for CHO cell lines whenproven efficacious. These became some of the components described inmore detail below as Part C.

TABLE 1 Comparison of original cell culture medium formulation (Part A67147) with modified cell culture medium formulation (modified Part A67411). Cell culture Cell culture medium containing medium containingModified 67411 (RM-230) 67147 (RM-003) (modified Part A) Component(Original Part A) at 1X, 2X, 3X, and 4X g/L Part A Part A OriginalPowder: JRH Source 16.45 g/L NA 1X concentration only Modified Part A:Special JRH Source NA 2.63, 5.26, 7.89, and 10.52 g/L Selectedcomponents without sodium bicarbonate, HEPES, and the mono- and di-basic sodium phosphates, the osmolarity regulator sodium chloride, thesurfactant Pluronic F-68, and the monosaccharide Glucose Part B Part B:Ferric Citrate JRH Stock solution 10 mL/L (0.5 mM) 10 mL/L (0.5 mM) PartC: Growth Factor, monosaccharide, energy source-amino acid: multiplelevels Recombinant hu Insulin 2-4 mg/L 4-13 mg/L Glucose 1.5-3.5 g/L To7.0 g/L max L-glutamine 0.292 g/L 0.584 g/L Buffers: Held to singleconcentration Sodium Bicarbonate 1.6 g/L 1.6 g/L HEPES NA 1.8 g/LNaH₂PO₄-H₂O NA 0.031 g/L Na₂HPO₄-7H₂O NA 0.436 g/L Osmolarity Regulator:Multiple levels per product line NaCl NA 0-6.5 g/L Hydrolysates:Multiple levels per product line Bacto TC Yeastolate NA 2.0-10.7 g/L BDPhytone (Soy) Peptone NA 0-6.92 g/L Primatone NA 2-8 g/LOther/Additional Agents Selective pressure (dhfr system): Multiplelevels per product line Methotrexate To required To requiredAmplification level Amplification level Surfactant-Shear protectant:Single level Pleuronic F-68 1.0 g/L 1.0 g/L Acid-Base for pH adjustmentNaOH As needed As needed HCl As needed As needed Solution targets FinalpH 7.2-7.4 7.1-7.3 Final Osmolarity 280-320 320-450Part A of Cell Culture Medium for CHO Cell Culture

As described above, Part A of the improved cell culture medium containeda modified version of a basal medium, i.e., PF-CHO media. PF-CHO media,(Catalog No. 67147) obtained from JRH, was modified by removing thebuffers sodium bicarbonate, HEPES, and the mono- and di-basic sodiumphosphates, the osmolarity regulator sodium chloride, the surfactantPluronic F-68, and the monosaccharide Glucose from Part A. Thesecomponents were added back to Part A in at various concentrationsdepending on the needs of the specific cell culture project. Thisallowed the buffer concentration to be held steady, and the surfactantconcentration to be kept below levels toxic to the cells, while allowingthe osmolarity and the carbon substrate levels to be manipulated forincreased growth of cells and increased antibody production. Once thesecomponents were separated from the original JRH Part A formulation, itfacilitated increases in the concentration of the remaining componentsin JRH Part A as seen in the modified cell culture medium (modified PFCHO—referred to as #67411 in Table 1), thus allowing for increased cellgrowth, and expression of antibody as determined by antibody titer.Individual components of the modified Part A powder (67411) formulation,including amino acids, vitamins, trace elements and other miscellaneouscomponent fractions were increased up to four times the concentration ofthe original formulation without having to change the volume of themedia. A comparison of the original Part A formulation using 67147 andthe modified formulation using 67411 is described above in Table 1.

By definition, the original JRH PF CHO basal medium (catalog #67147) wasa PF CHO basal medium without glutamine and without sodium bicarbonate.The original concentration of glucose in Part 1 was 1.5 mg/L.

Part B of Cell Culture Medium for CHO Cell Culture

The Part B component of the cell culture medium comprised a concentratedferric citrate solution, which was the same as the Part B component inPF-CHO media, (Catalog Nos. 67147) obtained from JRH, and was addedseparately.

Inorganic iron sources such as the ferric and ferrous salts,particularly ferric citrate and ferrous sulfate were added to the basalmedium, i.e., Part A or modified Part A. Though a small amount offerrous sulfate (0.2-0.8 mg/L) and ferric Nitrate Nonhydrate (0.025-0.11mg/L) was added, chelated salts, such as ferric citrate, were preferred.Ferric citrate was added in greater concentration as a liquidsupplement, and was included in the cell culture medium as PF-CHO PartB. Though the concentration of the other medium components could change,and have a greater range of concentration, ferric citrate was held to asingle concentration of 122 mg/L. This was due to the formation ofsuperoxides and free radicals causing cell damage, and the formation ofundesired compounds in the basal medium.

Part C of Cell Culture Medium for CHO Cell Culture

Part C of the cell culture medium comprised primarily of recombinantgrowth factors, buffers, an osmolarity regulator, an energy source, andhydrolysates. Additional compounds added to the cell culture medium thatare not included in the usual groupings of amino acids, vitamins andco-factors, inorganic salts and buffers, trace elements or minerals, inthe course of development are described in Table 1 above as “Part C”.With respect to Part C mentioned below in Table 1, it should be notedthat the ingredients identified in Part C may be added separately or incombination.

Recombinant Growth Factors

The peptide hormone Insulin, or alternatively a recombinant analog, wasadded to the cell culture medium in a concentration range of 4-13 mg/L.IGF-1 can also be substituted or supplemented to the Insulin added tothe cell culture medium at a concentration of 50-100 ng/L.

Osmolarity Regulator

The osmolarity in the various cell culture media was in the range of 260mOsm to 460 mOsm. Regulation of osmolarity was through salts especiallyNaCl, KCl, and KNO₃, though all of the amino acids and the hydrolysatescontribute considerably to changing osmolarity.

Energy Source

The most abundant monosaccharide in the medium was glucose (D-glucose)and was supplemented as needed. The starting concentration in the cellculture medium ranged from 3.5 to 7.0 g/L. Other sugars can also besupplemented as a metabolite or as a shear protectorant, these caninclude maltose, mannose, galactose, or fructose.

Hydrolysates

The hydrolysates are considered an additional source of free amino acidsalong with di- and tri-peptides.

pH Maintenance (Buffer)

Various buffers were used to maintain pH in the cell culture medium in arange of 6.5 to 7.5. The inorganic buffers (or buffer system) used inthe media included carbonates (NaHCO₃), chlorides (CaCl₂), sulphates(MgSO₄), and phosphates (NaH₂ PO₄ and Na₂HPO₄). Organic buffers alsoincludes Sodium Pyruvate (C₃H₃O₃Na) andN-[2-hydroxyethyl]piperazine-N′-[2-ethanesul-phonic acid] also known asHEPES.

Glutamine

Glutamine was also included in Part C, as it was omitted from bothoriginal Part A and modified Part A. Glutamine was included in Part C,e.g., 0.2 to 0.4 (0.29) g/L in the cell culture medium containingoriginal Part A and 0.3 to 0.7, e.g., 0.58, in the cell culture mediumcontaining modified Part A (see Table 1 above).

Other Components of Cell Culture Medium for CHO Cell Culture

The following provides additional components that may be added to thecell culture medium. It should be noted that additional components thatmay be added are not limited to the examples provided below.

Additional Peptides

Putrescine HCl salt, which aids in maintaining Endoplasmic reticulumstructure and growth specific to CHO cell lines, was added from 0.4 to1.65 mg/L to the Basal medium.

Glutathione (a tripeptide) was added in amounts ranging from 0.5 to 2.0mg/L. 2-Mercaptoethanol was also added to 3.6 mg/L, act as reducingagents in maintaining sulfhydryls and binds and transport various metalslike copper. It reduces dehydroascorbate and cystine and regeneratesascorbate and cysteine.

Methotrexate:

Methotrexate concentrations differed between product lines depending onthe final amplification levels to be achieved. Using a stock solution of2 mM concentration, the addition volumes and final concentrations are:0.250 mL/kg yields 500 nM final for both anti-IL-12 and anti-EPO-R, 0.5mL/kg yields 100 nM final for anti-IL-18, and finally 2.5 mL/kg yields5000 nM final for anti-TNF alpha.

Cell Protectant/Surfactant

The media described in this example was used to grow CHO cells insuspension in all scale reactors, flasks, and spinner flasks under bothagitation and sparging, which created larger shear forces. To minimizecellular damage a cell protectorant like a pluronic polyols,specifically Pluronic F-68 at a concentration of approximately 1 g/L ofmedium was added. Other shear mitigators including methyl cellulose atless than and equal to 1 g/L, and certain hydrolysates and plantextracts at varying concentrations up to gram quantities per liter werealso used.

Amino Acids

The cell culture medium had amount ranges of amino acids as describedherein. Two amino acids, Asparagine and glutamine, were added in greaterstarting concentrations than the other amino acids as they quicklybecome limiting nutrients during the course of CHO cell culture.Asparagine in particular is about 0.4 to 0.5 g/kg in the basal medium.

While asparagine was a component in Part A (including both original andmodified PF CHO), the concentration of asparagine was increased bysupplementing it into the cell culture medium.

The cell culture medium was capable of supporting CHO growth from verylow initial densities to over 1.0×10⁷ cells/mL for a number of daysdepending on the concentration of the components with purpose andcellular effect of the media desired. The CHO process additionallyincluded a growth phase and a production phase as described in latersections, and required different ranges of components. However the cellculture medium formulation proportions and identities remained the same.

Final Preparation of CHO Cell Media

Preparation of the improved cell culture medium required addition ofvarious components in a particular order with pH adjustments using abase or acid at particular times. Base in the form of NaOH was added asthe Part A basal concentration increased to aid in the dissolution ofthe amino acids in the formulation to a maximum pH of 10. The pH wasbrought down to minimum of 7.0 as the Hydrolysates were added using acidin the form of HCl. Further adjustments up to a particular pH wereachieved using either the NaOH or HCl solutions as needed.

The following examples describe cell culture media based on the abovefor the expression of various specific antibodies.

Example 1.2 Cell Culture Medium for Culturing CHO Cells ExpressingAnti-TNF Alpha Antibody

CHO cell lines expressing a fully human antibody to TNFα (i.e.,adalimumab; D2E7) were cultured in the cell culture media described inTable 2.

TABLE 2 Cell culture media for culturing CHO cells expressing fullyhuman anti-TNF alpha antibody AF-D2E7- AF-D2E7- AF and AY-D2E7- AY-D2E7-1XP 1XP AY-D2E7 2XP 2XP Media Components Specification # Component list:SR-250 SR-286 JRH Part A and Raw SR-248 growth + production SR-332SR-333 Part B Material growth MTX prod_3XP growth 1 growth 2 ABCComponents Added Final pH: 7.2 ± 0.1 7.2 ± 0.1 7.2 ± 0.1 7.2 ± 0.1 7.2 ±0.1 Final 280-320 280-320 370-390 320-360 320-360 Osmolarity: Part A-:unmodified-original- RM-003 16.45 16.45 NA NA NA commercially availableSpecial (Modified) Part A: salt-free RM-230 NA NA 7.89 g/kg 5.26 g/kg5.26 g/kg Part B: Ferric citrate: Chelated Iron RM-004 10 ml/kg 10 mL/kg10 mL/kg 10 mL/kg 10 mL/kg source Part C: rHu Insulin: RecombinantProtein SR-055 2.0 mL/kg 2.0 mL/kg 6.0 mL/kg 3.88 mL/kg 3.88 mL/kgGlucose regulator (4 mg/kg) (4 mg/kg) (12 mg/kg) (8 mg/kg) (8 mg/kg)Glucose, anhydrous: Carbon source RM-011 3.5 g/kg 3.5 g/kg 7.0 g/kg 7.0g/kg 7.0 g/kg L-Glutamine: Amino Acid and energy RM-071 0.292 g/kg 0.292g/kg 0.584 g/kg 0.876 g/kg 0.876 g/kg source NaH2PO4•H2O: Phosphatebuffer RM-200 Part A: Part A: 0.031 g/kg 0.031 g/kg 0.031 g/kg 0.031g/kg 0.031 g/kg Na2HPO₄•7H2O: Phosphate buffer RM-233 Part A: Part A:0.436 g/kg 0.436 g/kg 0.436 g/kg 0.436 g/kg 0.436 g/kg Bacto TCYeastolate: Yeast source RM-216 2.0 g/kg 2.0 g/kg 10.7 g/kg 4.0 g/kg 4.0g/kg hydrolysate Phytone Peptone: Plant-Soy Source RM-238 NA NA 6.92g/kg 2.6 g/kg 2.6 g/kg hydrolysate Sodium Bicarbonate: Buffer: CO2-pHRM-077 1.6 g/kg 1.6 g/kg 1.6 g/kg 1.6 g/kg 1.6 g/kg regulator HEPES:Organic buffer RM-090 NA NA 1.8 g/kg 1.8 g/kg 1.8 g/kg NaCl (Salt):Osmolarity regulator RM-174 Part A: Part A: 2.45 g/kg 2.67 g/kg 2.67g/kg 6.5 g/kg 6.5 g/kg Other components: L-Asparagine monohydrate: AminoRM-284 NA NA NA 0.45 g/kg 0.45 g/kg Acid Pluronic F-68 (Poloxamer 188,NF): RM-188 NA NA 1.0 g/kg 1.0 g/kg 1.0 g/kg Surfactant, CarrierMethotrexate: Selective in the CHO SR-133 2.50 mL/kg 2.50 mL/kg 2.50mL/kg 2.50 mL/kg 2.50 mL/kg amplification DHFR system NaOH, 2N: BaseSR-288 As needed As needed 5.67 mL/kg 3.5 mL/kg 3.5 mL/kg HCl, 2N: AcidSR-287 As needed As needed 2.5 mL/kg 2.91 mL/kg 2.91 mL/kg

Example 1.3 Media Composition for Culturing CHO Cells Expressing IL-12Antibody

The CHO cell line, expressing a fully human, anti IL-12 antibody wascultivated in a growth medium, described in Table 3.

TABLE 3 Media for culturing CHO cells expressing fully human anti-IL12antibody (ABT-874) ABT-874 ABT-874 ABT-874 ABT-874 ABT-874 ABT-874ABT-874 Specification # Raw SR-383 SR-352 SR-468 SR-351 Media Name,Material growth Production Feed Glu feed SR-274 SR-273 SR-272 Final pH:,6.5-6.9 6.5-6.9 6.5-6.9 Final Osmolarity: 265-282 265-282 265-282 PartA-: RM-003 NA NA NA NA 16.45 g/kg 16.45 g/kg 16.45 g/kg unmodified-original- commercially available Part A (Modified): RM-230 5.26 g/kg NANA NA NA NA NA salt-free Part A (Modified): RM-322 NA 7.89 g/kg 21.0g/kg 7.89 g/kg NA NA NA salt-free & reduced vitamins Part B: FerricRM-004 10 mL/kg 10 mL/kg NA 10 mL/kg 10 mL/kg 10 mL/kg 10 mL/kg citrate:Chelated Iron source Part C: Bovine SR-057 NA NA NA NA NA NA NATransferrin: Animal source Fe carrier rHu Insulin: SR-055 3.88 mL/kg 6.5mL/kg NA 6.5 mL/kg 2 mL/kg 2 mL/kg 2 mL/kg Recombinant (13 mg/kg) (4mg/L) (4 mg/L) (4 mg/L) Protein Glucose regulator Glucose, RM-011 7.0g/kg 7.0 g/kg 150 g/kg 200 g/kg 3.5 + 1.5 g/kg 3.5 + 1.5 g/kg 200 g/Lanhydrous: Carbon source L-Glutamine: RM-071 0.876 g/kg 0.584 g/kg NA0.584 g/kg 0.292 g/kg 0.292 g/kg 0.292 g/kg Amino Acid and energy sourceSodium RM-077 1.60 g/kg 1.60 g/kg NA 1.6 g/kg 1.6 g/kg 1.6 g/kg 1.6 g/kgBicarbonate: Buffer: CO2-pH regulator HEPES: Organic RM-090 1.80 g/kg1.80 g/kg NA 1.8 g/kg NA NA NA buffer NaCl (Salt): RM-174 2.675 g/kg2.45 g/kg NA 2.45 g/kg NA NA NA Osmolarity regulator NaH2PO4•H2O: RM-2000.031 g/kg 0.031 g/kg NA 0.031 g/kg NA NA NA Phosphate bufferNa2HPO₄•7H₂O: RM-233 0.436 g/kg 0.436 g/kg NA 0.436 g/kg NA NA NAPhosphate buffer Bacto TC RM-216 4.0 g/kg 10.7 g/kg 65.0 g/kg 10.7 g/kg2 g/kg 11 g/kg 8 g/kg Yeastolate: Yeast source hydrolysate PhytonePeptone: RM-238 2.579 g/kg 6.92 g/kg 41.0 g/kg 6.92 g/kg NA NA NAPlant-Soy Source hydrolysate Other components Pluronic F-68 RM-188 1.00g/kg 1.00 g/kg NA 1.0 mL/kg NA NA NA (Poloxamer 188, NF): Surfactant,Carrier L-Asparagine RM-284 0.450 g/kg NA 5.0 g/kg NA NA NA NAmonohydrate: Amino Acid Primatone: Beef- RM-149 NA NA NA NA NA NA NAAnimal Source hydrolysate Methotrexate: SR-133 0.250 mL/kg NA NA NA0.250 mL/kg 0.250 mL/kg 0.250 mL/kg Selective in the CHO amplificationDHFR system NaOH, 2N: Base SR-288 3.50 mL/kg 5.67 mL/kg As needed 5.67mL/kg As needed As needed As needed HCl, 2N: Acid SR-287 2.91 mL/kg 2.5mL/kg As needed 2.5 mL/kg As needed As needed As needed

With respect to the modified basal medium which is salt-free and hasreduced vitamins, referenced above, the vitamin amount is reduced onethird relative to unmodified basal medium, described above as RM-003, ormodified salt-free basal medium, described above as RM-230. Thus, areduced vitamin basal medium, as described above, has one third theamount of vitamins as RM-003 and RM-230. When used in amounts given inthe above table, the final concentration of vitamins in the media isreduced to a third, in contrast to having used RM-003 or RM-230. Itshould be noted, however, that if needed, production and feed mediumusing RM-230 may also be used.

Example 1.4 Media Composition for Culturing CHO Cells Expressing IL-18and EPO/R Antibodies

Table 4 provides a summary of the growth and production media used toexpress anti-IL18 and anti-EPO/R antibodies. Additional detailsregarding the media for expressing these antibodies can be found inTable 5 (anti-IL-18) and Table 6 (anti-EPO/R).

TABLE 4 Medium for culturing CHO cells expressing fully human anti-IL18and anti-EPO/R antibodies Anti-IL18- Anti-IL18 Anti-IL18 Anti-EPO/RAnti-EPO/R 2XP 3xP 4XP 1XP 3xP Media Components Specification #Component list: JRH Part A and Raw SR-371 SR-372 SR-382 SR-274 SR-286Part B Material growth production production growth production ABCComponents Added Final pH: 7.0 ± 0.1 6.9 ± 0.05 7.0 ± 1.0 7.2 ± 0.1 7.2± 0.1 Final 280-300 373-403 360-400 280-320 370-390 Osmolarity: Part A-:unmodified-original- RM-003 NA NA NA 16.45 NA commercially availableSpecial (Modified) Part A: salt- RM-230 5.26 g/kg 7.89 g/kg 10.52 g/kgNA 7.89 g/kg free Part B: Ferric citrate: Chelated RM-004 10 mL/kg 10mL/kg 10 mL/kg 10 mL/kg 10 mL/kg Iron source Part C: rHu Insulin:Recombinant SR-055 3.88 mL/kg 6.0 mL/kg 6.5 mL/kg 2.0 mL/kg 6.0 mL/kgProtein Glucose regulator (8 mg/kg) (12 mg/kg) (12 mg/kg) (4 mg/kg) (12mg/kg) Glucose, anhydrous: Carbon RM-011 7.0 g/kg 7.0 g/kg 7.0 g/kg 1.5g/kg 7.0 g/kg source L-Glutamine: Amino Acid and RM-071 0.876 g/kg 0.584g/kg 0.584 g/kg 0.292 g/kg 0.584 g/kg energy source Sodium Bicarbonate:Buffer: RM-077 1.6 g/kg 1.6 g/kg 1.6 g/kg 1.6 g/kg 1.6 g/kg CO2-pHregulator HEPES: Organic buffer RM-090 1.8 g/kg 1.8 g/kg 1.8 g/kg NA 1.8g/kg NaCl (Salt): Osmolarity regulator RM-174 2.67 g/kg 2.45 g/kg 2.45g/kg Part A: 2.45 g/kg 6.5 g/kg NaH2PO4•H2O: Phosphate RM-200 0.031 g/kg0.031 g/kg 0.031 g/kg Part A: 0.031 g/kg buffer 0.031 g/kg Na2HPO4•7H2O:Phosphate RM-233 0.436 g/kg 0.436 g/kg 0.436 g/kg Part A: 0.436 g/kgbuffer 0.436 g/kg Bacto TC Yeastolate: Yeast RM-216 4.0 g/kg 10.7 g/kg14.27 g/kg 2.0 g/kg 10.7 g/kg source hydrolysate Phytone Peptone:Plant-Soy RM-238 2.6 g/kg 6.92 g/kg 9.23 g/kg NA 6.92 g/kg Sourcehydrolysate Other: L-Asparagine monohydrate: RM-284 0.45 g/kg NA NA NANA Amino Acid Pluronic F-68 (Poloxamer 188, RM-188 1.0 g/kg 1.0 g/kg 1.0g/kg NA 1.0 g/kg NF): Surfactant, Carrier Methotrexate: Selective in theSR-133 0.05 mL/kg 0.05 mL/kg 0.05 mL/kg 0.25 mL/kg 2.50 mL/kg CHOamplification DHFR system NaOH, 2N: Base SR-288 As needed As needed Asneeded As needed 5.67 mL/kg HCl, 2N: Acid SR-287 As needed As needed Asneeded As needed 2.5 mL/kg

The IL-18 expressing CHO cell line was cultivated in a growth medium 2×P(SR-371), and later produced antibody in a production medium 3×P(SR-372) for a final titer of approximately 1 g/L. The high titerprocess used 4×P (SR-382) as the production medium to reach a finaltiter of approximately 2 g/L. The production medium used for anti-EPO/Rproduction was identical to SR-286, but a 1×P medium (SR-274) was usedfor cell growth. All media are described in Tables 4 and 5.

Example 1.5 Cell Culture Processes for Producing Antibodies in MammalianCells

The medium disclosed above was also developed into two productionplatforms used in two projects for culturing mammalian, i.e., CHO,cells. The first platform was developed using the similar mediumcomposition as described in the modified production medium described inTables 2-4 above, with the only difference being in the temperature usedfor cell culture. This platform was used for the production of anti-IL18antibody as well as the production of anti-erythropoietin receptor(anti-EPO/R). The second medium platform further strengthening thenutritional components, was used for high-titer anti-IL18 production, toachieve higher volumetric antibody productivity.

All antibodies, including anti-IL-12, anti-IL-18 and anti-EPO/Rantibodies were fully human IgG1 antibodies expressed by transfecteddhfr(−) CHO cell lines as described previously. These cell lines werecultivated in suspension and without the aid of bovine source serum orother animal source materials.

To produce anti-IL 18 antibodies, the anti-IL 18 expressing CHO cellline, was cultivated in a growth medium, herein referred to as SR-371.Medium SR-371 was used to support higher cell productivity with moderatecell growth. Once the cell density reached the transfer criteria, thecells were transferred into the production medium (SR-372) to start theproduction stage.

TABLE 5 Compositions of culture media in Anti-IL18 Process A. GrowthProduction medium Medium Component SR-371 SR-372 PFCHO Part A, specialsalt-free formulation 5.26 g/L 7.89 g/L PFCHO Part B (ferric citratestock solution) 10 mL/L 10 mL/L Recombinant human insulin 7.76 mg/L 13mg/L Dextrose, anhydrous 7.0 g/L 7.0 g/L L-glutamine 0.876 g/L 0.584 g/LSodium bicarbonate 1.6 g/L 1.6 g/L HEPES 1.8 g/L 1.8 g/L NaCl 2.67 g/L2.45 g/L Pluronic F-68 (Poloxamer 188, NF) 1.0 g/L 1.0 g/L NaH₂PO₄•H₂00.031 g/L 0.031 g/L Na₂HPO4•7H₂O 0.436 g/L 0.436 g/L Bacto TC Yeastolate4.0 g/L 10.7 g/L Phytone Peptone 2.579 g/L 6.92 g/L Methotrexate, 2 mM0.05 mL/L 0.05 mL/L NaOH, 2N 3.5 mL/L 5.67 mL/L HCl, 2N 2.91 g/L 2.5mL/L Final pH 7.10-7.20 7.10-7.20 Final osmolality (mOsmo/kg) 373-403373-403 Growth medium SR-371 was used in see train and seed reactors.Production medium SR-372 was used in 3000-liter production bioreactor.

The temperature was maintained at 35° C. throughout the culture.Additional 4 g/L of glucose was added when the cell culture glucoselevel was below 2 g/L.

A similar process was also employed for the anti-EPO/R antibodyproduction. However, a leaner medium (SR-274) was used for cell growthin the seed train. Medium SR-286, the same medium used for Humiraproduction, was used at the production stage for anti-EPO/R antibody(Table 6). Growth medium SR-274 was used in seed train and seedreactors. Production medium SR-286 was used in 3000-liter productionbioreactor.

TABLE 6 Compositions of culture media in Anti-EPO/R Process GrowthProduction medium medium Component SR-274 SR-286 PFCHO Part A, RM-00316.45 g/Kg N/A PFCHO Part A, RM-230 (salt-free) N/A 7.89 g/Kg PFCHO PartB (ferric 10 mL/Kg 10 mL/Kg citrate stock solution) Recombinant humaninsulin 4 mg/Kg 13 mg/Kg Dextrose, anhydrous 1.5 g/Kg 7.0 g/KgL-glutamine 0.292 g/Kg 0.584 g/Kg Sodium bicarbonate 1.6 g/Kg 1.6 g/KgHEPES N/A 1.8 g/Kg NaCl N/A 2.45 g/Kg Pluronic F-68 (Poloxamer 188, NF)N/A 1.0 g/Kg NaH₂PO₄•H₂0 N/A 0.031 g/Kg Na₂HPO4•7H₂O N/A 0.436 g/KgBacto TC yeastolate 2.0 g/Kg 10.7 g/Kg Phytone peptone N/A 6.92 g/KgMethotrexate, 2 mM 0.25 mL/Kg N/A NaOH, 2N As needed 5.67 mL/Kg HCl, 2NAs needed 2.5 mL/Kg Final pH 7.20 ± 0.10 7.15 ± 0.05 Final osmolality(mOsmo/kg) 320 ± 20  388 ± 15  Growth medium SR-274 was used in spinnerflasks, Wave bag, 100 L seed bioreactor Z-4605 and the initial stage of575 L culture in the 3000 L production bioreactor Z-3600. Productionmedium SR-286 was used in 3000 L production bioreactor Z-3600 only.

The production media SR-286 and SR-372 consisted of similar mediumcomponents as listed in Tables 5 and 6 but with different level of MTX(0 nM for SR-286 and 100 nM for SR-372).

An improved process was developed for anti-IL18 production to obtainhigher productivity. This new process, Process B, introduced a newmedium for extending cell culture longevity and increasing antibodyvolumetric productivity. The production medium (SR-382) was differentfrom the previous production media on the amount of nutrients used atthe production stage. The full composition of SR-382 is described inTable 7. In the new process for anti-IL18 production, although cellswere still cultivated in medium SR-371 during the seed train, mediumSR-372 was used in the seed bioreactor one-step before the productionstage. Cells were then cultivated in medium SR-382 at the productionstage with a temperature shift from 35° C. to 33° C. to prolong the cellculture longevity therefore extending the effects of medium SR-382 tothe cells.

Growth medium SR-371 was used in spinner flasks, Wave bag, and 100 literseed bioreactor. Short-fill medium SR-372 was used in the initial stageof 575-liter culture in the 3000-liter production bioreactor. Productionmedium SR-382 was used in the 3000-liter production bioreactor only.

TABLE 7 Compositions of culture media in Anti-IL18 Process B GrowthShort-fill Production medium medium medium Component SR-371 SR-372SR-382 PFCHO Part A, special 5.26 g/L 7.89 g/L 10.52 g/L salt-freeformulation PFCHO Part B (ferric 10 mL/L 10 mL/L 10 mL/L citrate stocksolution) Recombinant human 7.76 mg/L 13 mg/L 13 mL/L insulin Dextrose,anhydrous 7.0 g/L 7.0 g/L 7.0 g/L L-glutamine 0.876 g/L 0.584 g/L 0.584g/L Sodium bicarbonate 1.6 g/L 1.6 g/L 1.6 g/L HEPES 1.8 g/L 1.8 g/L 1.8g/L NaCl 2.67 g/L 2.45 g/L 0 g/L Pluronic F-68 1.0 g/L 1.0 g/L 1.0 g/L(Poloxamer 188, NF) NaH₂PO₄•H₂0 0.031 g/L 0.031 g/L 0.031 g/LNa₂HPO4•7H₂O 0.436 g/L 0.436 g/L 0.436 g/L Bacto TC Yeastolate 4.0 g/L10.7 g/L 14.27 g/L Phytone Peptone 2.579 g/L 6.92 g/L 9.23 g/LMethotrexate, 2 mM 0.05 mL/L 0.05 mL/L 0.05 mL/L NaOH, 2N 3.5 mL/L 5.67mL/L 8.95 mL/L HCl, 2N 2.91 g/L 2.5 mL/L 4.1 mL/kg Final pH 7.1-7.27.1-7.2 7.1-7.2 Final osmolality 373-403 373-403 373-403 (mOsm/kg)

Nutrients were enriched in this medium to further provide the energysources and building components for CHO cell growth and antibodyproduction. In the process B, although cells were still cultivated inmedium SR-371 during the seed train, medium SR-372 was used in the seedbioreactor one-step before the production stage. Cells were thencultivated in medium SR-382 at the production stage with a temperatureshift from 35° C. to 32° C. to prolong the cell culture longevitytherefore extending the effects of medium SR-382 to the cells.

Process A: Performance of Anti-IL 18 Cells and Anti-EPO/R Cells inMedium SR-372 and Medium SR-286

Anti-IL18 expressing cells were cultivated in medium SR-371 with 100 nMMTX to accumulate cell mass for the production stage. Medium SR-371 wasused to support higher cell productivity with moderate cell growth.Table 8 shows a representative production growth profile of Anti-IL18producing CHO cells in the 3000 L production bioreactor. Using thisprocess (Process A) with medium SR-372, a final titer up to 1 g/L can beobtained.

TABLE 8 Production of Anti-IL18 antibody in Medium 372 (Process A)Bench-scale 3000 L process (n = 5) Process (n = 6) Measurable ResultsTemp = 35° C. Temp = 35° C. Maximum Cell Density 8.68 9.2 [10⁶ viablecells/ml] Duration to 50% Viability 11 11 [Days] Cell SpecificProductivity 20.5 17.6 [pg/cell-day] Volumetric Productivity 98.8 81.8to Harvest @ 50% Viable [mg/L-day] Titer @ 50% Viable 1004 900 [mg/L]

Medium 286, which shares same formulation as Medium 372 except for theMTX level, was used for anti-EPO/R production. Although usually a lowercell density is obtained at the production stage, a higher productivitywas reached to enable the cells producing up to 1.8 g/L of antibody at3000 L scale by using medium SR-286 as the production medium. Reasonablecell growth was observed as summarized in Table 9. Bench-scale resultsas well as results from 3000 L run demonstrated that this mediumincreased cell specific productivity and a final titer up to 1.9 g/L wasobserved. These results show that media with the similar formulation(SR-372 and SR-286 in Tables 1 and 2) support good cell growth and highantibody production rates in large scale CHO cell culture.

TABLE 9 Production of Anti-EPO/R antibody in Medium 286 Bench-scale 3000L process (n = 2) Process (n = 1) Measurable Results Temp = 35° C. Temp= 35° C. Maximum Cell Density 4.50 4.80 [10⁶ viable cells/ml] Durationto 50% Viability 13 13 [Days] Cell Specific Productivity 38.0 53.2[pg/cell-day] Volumetric Productivity 114.4 146.1 to Harvest @ 50%Viable [mg/L-day] Titer @ 50% Viable 1487 1900 [mg/L]Process B: Performance of Anti-IL 18 Cells in Process B with MediumSR-382

Medium SR-382 was the most enriched medium used in the extended batchprocess for anti-IL-18 antibody production. Process B includes usingmedium SR-371 in the seed train and SR-372 in the seed bioreactor beforethe production stage, or the short-fill stage. The cell growth wasmoderate compared to the cell growth in medium SR-372 at the productionstage. However, with the temperature shift, a final titer up to 2.5 g/Lwas obtained using Medium SR-382.

Medium SR-382 was developed based on the study showing that the cellspecific productivity of anti-IL18 expressing cells proportionallyincreased with increasing nutrients in the production media. MediumSR-382 was the optimal medium which provided the balance between cellgrowth and final titer increase. Although the maximum cell density onlyreached 5.9×10⁶ cells/mL, the cell specific productivity increasedtwo-fold. Combining with temperature shift to prolong the cell cultureduration, a final titer up to 2.2 g/L was achieved as shown in Table 10.

TABLE 10 Production of Anti-IL18 antibody in Medium SR-382 (Process B)Bench-scale 3000 L process (n = 1) Process (n = 1) Measurable ResultsTemp = 35-33° C. Temp = 35-33° C. Maximum Cell Density 7.85 5.90 [10⁶viable cells/ml] Duration to 50% Viability 12 13 [Days] Cell SpecificProductivity 32.0 42.1 [pg/cell-day] Volumetric Productivity 181.1 191.8to Harvest @ 50% Viable [mg/L-day] Titer @ 50% Viable 2173 2110 [mg/L]

Example 2 Improved Fed Batch Process and Feed Solutions for ExpressingAntibodies

Spent medium analysis of bioreactors run in batch mode showed depletionof certain amino acids. This finding also suggested depletion of othermedium components, even if not measured, which could lead to additionalnutritional deficiencies. In other to compensate for these potentialdeficiencies, solutions of nutrients were added. In the engineeringfield, this approach is generally referred to as fed-batch.

For an operational point of view it is convenient to use concentratedfeed solutions. The following examples describe the addition of highlyconcentrated solutions of the chemically defined basal medium (PFCHO,Catalog #67411-50L) and of complex hydrolyzates, e.g., yeastolate andphytone. It was determine that this pair of hydrolyzates exhibited aproductivity increase synergistic effect related to their concentrationratio.

Example 2.1 Adalimumab Fed Batch Process

The initial adalimumab (Humira/D2E7) process consisted of a 3 dayprocess in which media was removed and replenished eight consecutivetimes. An improved fed batch process was developed by replacing in themedium the hydrolyzate Primatone with the hydrolyzate Yeastolate and byusing new reactor parameters. The improved fed batch process lastedapproximately 12 days.

The productivity of the initial batch process was further improved byre-formulating the basal medium, PFCHO, and adding a new hydrolyzate,i.e., Phytone. This hydrolyzate containing media formulation was used inthe process referred to above as SR-286 (see Table 2). Reactor operatingparameters were also investigated which resulted in the identificationof an optimal temperature to run the entire adalimumab productionprocess.

Analysis of samples taken daily from the reactor experiments highlightedsome potential nutritional deficiencies. For the case of the adalimumabbatch process, the potential nutritional deficiency was addressed byfeeding a 25× concentrated PFCHO solution and a 33× solution of thehydrolyzates yeastolate and phytone. The hydrolyzates are complexcomponents that exhibit a synergistic effect related to theirconcentration ratio. This ratio was maintained in a highly concentrated33× version.

Experimental Plan

The goal of the experiment was to compare the new fed batch process totwo batch control processes (temperature shift 37-*33° C. vs. constanttemperature 35° C.). The fed-batch modifications were:

-   1. 25× basal media enrichment (PFCHO solution) fed based on amino    acid deficiencies.-   2. 33× hydrolyzate enrichment solution fed at intervals so that the    osmolarity of the media never exceeded 440 mOsm (a condition that    results in reduced cell growth and viability)-   3. The reactor temperature set-point to be 35° C. throughout.    The controls for this experiment were:-   a) An identical reactor operating with the current media (SR-286)    and under control batch process parameters (control conditions    included reactors running SR-286 medium with a temperature shift and    linear pH ramp), designated as control #1; and-   b) An identical reactor operating with the current media (SR-286)    and under all the current batch process parameters except for an    operating temperature of 35° C. throughout, designated as control    #2.    Materials    Braun ED reactors with a working volume of 13 L    Pilot Plant Inoculum AFI915A using Working Cell Bank WCB970513-6    3XP11Y7P Basal media solution (SR-286)    Basal Media Enrichment Solution (25×) (PF CHO solution)    Hydrolysate Enrichment Solution (33×)    Glucose Feed Shot (200 g/L) (Glucose solution)    0.5N Sodium Hydroxide Solution for pH control    Solution Preparation:-   1. Production Medium (see SR-286 Solution Record described above in    Table 2)-   2. 2 kg of PFCHO Enrichment Solution (25×) (Basal enrichment    solution): Prepared in the following order, under constant stirring    and allowing mixing for 10 minutes after each addition step:

Component Mass [g] Notes MilliQ H₂O 1500 PFCHO 131.5 10N NaOH 49 mLUntil pH 10 Asparagine 15 pH will drop to ~9.73 Glucose 100 pH will dropto ~9.71 MilliQ H₂O As required Bring weight to 2000 g, pH ~9.70,osmolarity ~1480 mOsm Filter with 0.2μ PES filter membrane Store at 4°C. Every addition of 1% of initial volume of the above solution willincrease: a) PFCHO concentration by 0.25x compared to original 3xconcentration b) Asparagine by 75 mg · L⁻¹ c) Glucose concentration by0.5 g · L⁻¹ d) Osmolarity by 10 mOsm e) pH by ~0.10 pH units

-   3. 1 Kg Hydrolysate Enrichment Solution (33×):    -   Prepared in the following order, under constant stirring and        allowing mixing for 10 minutes after each addition step:

Component Mass [g] Notes MilliQ H₂O 500 TC Yeastolate 265 PhytonePeptone 165 MilliQ H₂O As required Bring weight to 1000 g Filter with0.2μ PES filter membrane Store at 4° C. Note: Every addition of 1% ofinitial volume of the above solution will increase: a) TC Yeastolateconcentration by 2.65 g/L (0.33x) compared to original batchconcentration. b) Phytone Peptone concentration by 1.65 g/L (0.33x)compared to original batch concentration.Methods:

Reactor Operation:

-   -   To inoculate the reactor, a vial was thawed and expanded        following the Humira seed train process description. After        growing in the reactor, the reactor was drained down to 3.62 L        to simulate the shortfill stage. Then reactor was topped off to        13 L level with production media (SR-286).

Reactors were operated with the following parameters:

-   -   a) Agitation 70 RPM    -   b) Temperature 35° C.    -   c) pH linear ramp started from pH 7.16 to pH 6.90 over a 72 hour        period    -   d) Dissolved oxygen 30%    -   e) Reactors were fed 195 g of a 200 g/L glucose solution when        glucose level was under 2.0 g·L⁻¹        Feeding Schedule:

The following represents the feeding schedule for adding the additionalnutrients, i.e., supplemental basal medium, and hydrolyzates to theadalimumab batch.

TABLE 11 Feed schedule adalimumab fed batch process Feed Amounts [g] Day25x PFCHO 33x Hydrolyzates 0-3 4 130 130 5 6 260 7 130, glucose 8 9 260,glucose 10  11  130 12-13Results:

Results (as well as projected improvements) comparing control processes#1 and #2 to the improved fed batch process are described in Tables 12and 13. As shown in Table 12, adalimumab productivity increased with theaddition of enhanced enriched basal media and hydrolyzate enrichmentsolution using the improved fed batch process under constanttemperature.

TABLE 12 Comparison of adalimumab fed batch processes Control #2 Control#1 (3000 L Fed-Batch (3000 L Process) Process) Experiment Temp = Temp =Temp = Measurable Results 37° C. ↓ 33° C. 35° C. 35° C. Maximum CellDensity 3.63 4.45 4.41 [10⁶ viable cells/ml] Duration to 50% Viability13 10 12 [Days] Cell Specific Productivity 42.5 46.7 61.4 [pg/cell-day]Volumetric Productivity 98 114 163 to Harvest @ 50% Viable [mg/L-day]Titer @ 50% Viable 1322 1178 1979 [mg/L]

TABLE 13 Comparison of projected results using adalimumab fed batchprocesses Control #2 Control #1 (3000 L Fed-Batch (3000 L Process)Process) Experiment Temp = Temp = Temp = Projected outcome 37° C. ↓ 33°C. 35° C. 35° C. Harvests per Year 22 28 24 [Allowing 3 day turn around]Yearly Product Yield 75.6 85.7 123.4 (Based on 2600 L Harvest) [Kg/year]Increased Yearly Yield 100% 114% 163% (control = 100%)

Example 2.2 ABT-874 Fed Batch Process

As for the case of the improved fed batch process for adalimumabmentioned above, analysis of samples taken daily from the reactorrunning ABT-874 in batch mode highlighted amino acid depletion. Again,this deficiency was addressed by using a 25×PFCHO solution and theconcentrated 33× hydrolyzate solution.

The batch process for ABT-874 was originally developed for D2E7(adalimumab) by replacing the hydrolyzate in the media and byintroducing new reactor parameters. Furthermore, as in the case of D2E7(adalimumab), the basal media was reformulated and a new hydrolyzate wasadded. This media formulation is used in the current D2E7 process asSR-286 (see Table 2 above).

Experimental Plan:

The goal of the experiment was to compare a control batch process to thefollowing fed-batch conditions, both beginning on the time when aminoacid depletion had been previously identified:

-   a) Feed alternatively basal media enrichment 25×PFCHO and 33×    hydrolyzate enrichment solutions-   b) Feed daily both basal media enrichment 25×PFCHO and 33×    hydrolyzate enrichment solutions    The control for this experiment included an identical reactor    operating with the current media (SR-286) and under the control    batch process parameters (SR-286 medium, with a temperature shift    and linear pH ramp).    Materials:    -   Braun ED reactors with a working volume of 13 L    -   Working Cell Bank W990107-J695    -   3XP11Y7P Basal media solution (SR-286-111899-1)    -   PFCHO-0-500-HG2Y Growth Media    -   Basal Media Enrichment Solution (25×)    -   Hydrolysate Enrichment Solution (33×)    -   0.5N Sodium Hydroxide Solution        Solution Preparation:-   1. Production medium (see SR-286 Solution Record)-   2. 25×PFCHO Solution (basal enrichment solution: described in    previous example; with the exception that 462 g of glucose solution    were used instead of glucose powder, thus the final weight was 2170    g).

Note: Every addition of 1% of initial volume of the above solution willincrease:

a) PFCHO concentration by 0.25× compared to original 3× concentration

b) Asparagine by 75 mg·L⁻¹

c) Glucose concentration by 2.1 g·L⁻¹

-   -   pH by ˜0.10 pH units

-   3. 33× Hydrolysate Solution (Described in the previous example with    the addition of 2.40 g·L⁻¹ of glucose)    Note: addition of 1% of initial volume of the above solution will    increase:    -   a) TC Yeastolate concentration by 2.65 g/L (0.33×) compared to        original batch concentration.    -   b) Phytone Peptone concentration by 1.65 g/L (0.33×) compared to        original batch concentration.        Method:

To inoculate the reactor, a vial was thawed and expanded following theABT-874 seed train process description. After growing in the reactor,the reactor was drained down to 4.06 Liters (run designation B9013-ED2and B9014-ED3, as described in Table 13) to simulate shortfill. Thereactor was then topped off to 13 L level with regular production media(SR-286)

Reactors were operated according to the following parameters:

a) Agitation 70 RPM

b) Temperature 33° C.

c) pH 6.90

d) Dissolved oxygen 40%

TABLE 13 Feeding Schedule for ABT-874 Feed Amounts [g] Alternate FeedingSchedule Daily Feeding Schedule Run Designation B9013-ED2 RunDesignation B9014-ED3 25x PFCHO 33x Hydrolyzates 25x PFCHO 33xHydrolyzates 0-4 5 130 65 65 6 130 65 65 7 130 65 65 8 130 65 65 9 13065 65 10  130 65 65 11  130 65 65 12  130 65 65 13  130 65 65 14-15Results

Results comparing the improved fed batch processes are described belowin Tables 14 and 15.

TABLE 14 Fed batch process results Alternate Daily Control Fed-BatchFed-Batch (1000 L Process) Experiment Experiment Parameter Temp = 33° C.Temp = 33° C. Temp = 33° C. Maximum 3.79 5.39 4.15 Cell Density [10⁶cell/ml] Duration to 14 to 76% 15 15 50% Viability [Days] Cell Specific71 83 82 Productivity [pg/cell-day] Volumetric 188 281 212 Productivityto Harvest @ 50% Viable [mg/L-day] Titer @ 2505 @ 76% 3995 3033 50%Viable [mg/L]

TABLE 15 Fed batch process results Alternate Daily Control Fed-BatchFed-Batch (1000 L Process) Experiment Experiment Projected Outcome Temp= 33° C. Temp = 33° C. Temp = 33° C. Harvests per Year  21  20  20(Allowing 3 day turnaround) Yearly Product 137 208 158 Yield (Based on2600 L Harvest) [Kg/year] Increased 100% 152% 115% Yearly Yield (control= 100%)

Example 3 Stable Combination Feed Solutions for Increasing theVolumetric Productivity of Fed-Batch Culture

The following examples describe a novel approach to formulate stablehigh concentration feed solutions that include two hydrolyzates, atleast one amino acid other than glutamine, a sugar, and a chemicallydefined medium base. The resulting feed solutions are capable ofincreasing the volumetric production of mammalian cell lines producingrecombinant proteins. Finally, an accelerated method for fed-batchprocess development based on feedback control of the glucoseconcentration is proposed.

Materials and Methods

Combination feeds contained the hydrolyzates Bacto TC yeastolate,(RM-216) (BD Difco 255771) and phytone peptone, (BD Difco 2922450) plusglucose, L-asparagine monohydrate (Sigma-Aldrich) a reduced version ofDMEM/F12 (NaCl, phosphate salts, pH indicators and other non-essentialcomponents were removed; Invitrogen 12500) or Ex-Cell PFCHO (A)-S1(modified deficient) w/o glutamine, w/o NaHCO, (JRH Biosciences67411-500L35470).

For the preparation of solutions, water was filtered using a MilliporeMilli-Q PF with a PMQ004D2 filtration pack. Materials were dissolved inthe specified mass of water using a bench top magnetic stirrer. Aftereach component was added, complete dissolution was visually verifiedbefore the next component was incorporated.

When applicable, turbidity was quantified using a HACH 2100P portableturbidimeter (Hach Co. Loveland, Colo.). The human eye threshold fordetecting turbidity is around 15 NTUs (Nephelometric Turbidity Units).

Bioreactor experiments were carried in 3 L Applikon bioreactors at anoperating volume of 1.5 L with pH, temperature, agitation and oxygencontrol via cascading air and oxygen flow. Cell counting was done with aCedex (Innovatis AG, Bielefeld, Germany). Glucose, lactate weredetermined using a YSI 2700 (YSI Inc., Yellow Springs, Ohio) and in somecases also additional metabolites with a Nova Bioprofile 400 (NovaBiomedical Corp., Waltham, Mass.). The equilibrium partial pressure ofoxygen (pO₂), carbon dioxide (pCO₂), and pH was verified using an ABL 5Blood Gas Analyzer (Radiometer A/S, Copenhagen, Brønshøj, Denmark).

Example 3.1 Preparation of Stable Combination Feeds Using PFCHO as BasalMedium

A single, high concentration feed facilitates cell culture fed-batchmanufacturing, because it reduces the volume and the number of additionsrequired. This is however, complicated due to the fact that PFCHO powdermust be dissolved at pH values above 9.00. Furthermore, hydrolyzates aresoluble under neutral pH conditions. Thus, trying to simply mix bothcomponents at neutral or high pH values will lead to (non dissolved)powder suspensions. As reported in the literature, “Fully optimizedfeeds often exist as two or more separate solutions that support morethan one rate of introduction and feed pH (e.g., for reasons ofsolubility)” [1].

Combination Feed Stability Experiment

It was determined that the addition of hydrolysates allowed PFCHOconcentrations to remain stable for longer periods, as described in thefollowing table. The component amounts of 20 g Kg⁻¹ PFCHO, 7.5 g Kg⁻¹asparagine, 21 g Kg⁻¹ glucose, 22 g Kg⁻¹ yeastolate, and 14 g Kg⁻¹phytone were added in order to about 700 g of water and water was addedat the end to achieve a final weight of 1 Kg. Solutions were mixed andthen brought down to target pH with HCL 2.0 N. In the following table,turbidity was determined by visual observation with the naked eye.

TABLE 16 Effect of final pH on different combination feed formulationsFinal solution pH Formulation 6.75 7.00 7.25 7.50 7.75 1 PFCHO,Asparagine N/A N/A N/A Completely and glucose dissolved precipitated atpH 10.0 before 4 hrs 2 Same as above (1) to N/A N/A Final N/A N/A pH7.75, then add pH phytone and yeastolate 7.3 3 Same as above (2), Slightdegree of turbidity within 30-60 then pH to 6.75, 7.00, min; yetresulting solution remains stable 7.25, 7.50, 7.75

As seen in Table 16, the least turbid were 2) and 3) pH 6.75 and 7.25.Note that both 2) and 3) did not become turbid even after almost 24 hrs.Based on these results, it was clear that the hydrolyzates stabilizePFCHO in solution, as low turbidity was achieved.

Given that the hydrolyzates stabilize the resulting mixture, phytone andyeastolate could be added to the PFCHO solution at pH 10.0; then thewhole mixture be brought down to target pH. This order of addition wouldremove the unstable step of holding the PFCHO solution at pH valuesunder 8.0, particularly vulnerable when mixing larger volumes.

To test the previous hypothesis we tested this new order of additionwith a formulation containing 20 and 7.5 g·Kg⁻¹ of PFCHO and asparagine,respectively. The resulting solution, X-1, was divided and brought downto pH 7.0, 6.75, 6.5 and 6.25. These solutions proved stable, as can beobserved in the following graph:

TABLE 17 Turbidity profiles (NTU) of formulation X-1 at different pHvalues pH Time [hrs] 6.25 6.50 6.75 7.00 2.25 n/a 8.44 9.86 11.32 4.008.77 7.8 9.25 10.70 6.00 7.02 5.71 6.28 4.25

After three hours, the least turbid solution was pH 6.50. The apparentdecrease in turbidity, in particular for pH 7.0, was due to settling ofsome minute particles.

Glucose as a Stabilizer

A formulation (see Table 18) with 200 g Kg⁻¹ glucose added as astabilizer was tested.

TABLE 18 Diluted D2E7 feed solution _(MilliQ)H₂O 750.0 g · Kg⁻¹ Waterwas in excess Glucose 200.0 As potential stabilizer PFCHO 20.0 + NaOH10N → pH 10.00 Asparagine 2.29 Yeastolate 15.7 Phytone 10.0 + HCl 5N →pH 6.75-7.50 Components were weighted and added in order. Initial massof water should be reduced to achieve 1 Kg final mass.

This solution proved to be stable for several hours for a range of pHvalues, as can be observed in the following table:

TABLE 19 Turbidity readings (NTU) of combination feed as a function oftime and pH. pH Time [hrs] 6.72 7.00 7.22 7.50 0.25 3.48 3.75 3.95 5.832.50 3.07 3.13 3.42 3.74 8.50 2.98 2.95 2.99 3.15

The modified combination feed solutions comprising glucose were used toexpress two different antibodies, i.e., adalimumab (D2E7) and anti-IL-18antibody ABT-325.

Combination Feed Solution for Stable D2E7 Production

If the volume of formulation added to the bioreactor is based on thelowest concentrated component (i.e. PFCHO), very large amounts of feedwould need to be added to match what has been evaluated individually.Therefore, higher concentrations were the next step in developing aneffective feeding formulation. This solution is referred to as the D2E7combination feed solution and is described in the following table.

TABLE 20 D2E7 combination feed solution _(MilliQ)H₂O 750.0 g · Kg⁻¹Glucose 150.0 PFCHO 27.0 + NaOH 10N → pH 10.00 Asparagine 3.1 Yeastolate21.2 Phytone 13.5 + HCl 5N → pH 6.75 Components were weighted and addedin order. Initial mass of water should be reduced to achieve 1 Kg finalmass.

Formulations with 200, 150 and 100 g Kg⁻¹ glucose were tested. As can beseen in the following table, these solutions also had a stable turbidityfor several hours. Table 21 shows that the addition of glucose reducedthe turbidity of the solution.

TABLE 21 Turbidity time profiles for the D2E7 combination feed solutionas a function of glucose concentration. Glucose [g/Kg combination feed]Time [hrs] 100 150 200 0 n/a 9.23 7.31 1 13.80 8.77 n/a 2 13.20 9.33 n/a3 12.80 10.30 n/a 4 12.80 10.70 5.98 7 n/a n/a 6.80

As shown in Table 21, increasing the glucose level decreased turbidityof the solution. These different formulations, based on their turbiditylevels were regarded as acceptable for filtration experiments.

Combination Feed Solution for Stable ABT-325 Production

To obtain a stable ABT-325 combination feed, 50 L of the currentformulation was prepared according to the method used for the D2E7combination feed, as shown in Table 22.

TABLE 22 ABT-325 combination feed solution _(MilliQ)H₂O 750.0 g · Kg⁻¹Glucose 150.0 PFCHO 21.0 + NaOH 10N → pH 10.00 Asparagine 5.0 Yeastolate65.0 Phytone 40.0 + HCl 5N → pH 6.75 Components were weighted and addedin order. Initial mass of water should be reduced to achieve 1 Kg finalmass.

Upon preparation, the solution maintained a turbidity level ofapproximately 20-30 NTUs, as can be observed in the following table:

TABLE 23 Turbidity of ABT-325 Combination feed Turbidity Time [hrs[[NTUs] 0.00 41.8 1.00 28.7 1.50 19.8 2.00 20.0 3.50 14.5

50 L of an ABT-325 formulation combination feed solution was preparedaccording to the D2E7 method to test both the scalability andapplicability of the preparation method. As shown above, the solutionremained stable for four hours.

It should be noted that the PF CHO medium referred to in the aboveexample corresponds to the modified PF CHO (modified Part A) referred toin the cell culture medium of Example 1.

Example 3.2 Preparation of Stable Combination Feeds Using DMEM-F12 asBasal Medium

As described in the previous example, it was possible to manufacture astable combination feed solution with PFCHO and two hydrolyzates, aswell as glucose. The following example demonstrates that thismethodology may be applied to any basal feed formulation and leads to astable combination feed solution.

DMEM-F12, a medium formulation that is publicly available, was modifiedto make it compatible with combination feed preparation, denominatedhere as DMEM-F12m. The following components were removed: NaCl, NaHCO₃,NaH₂PO₄.H₂O, Na₂HPO₄, D-Glucose, HEPES, Na•Hypoxanthine, Phenol red,L-glutamine and thymidine. Combinations feeds matching the D2E7 andABT-325 feed formulations were prepared according to the methodologydescribed in examples 3.0 and 3.1. The final components and formulationsequence is shown in the following table:

TABLE 24 DMEM-F12 combination feeds Feed I Feed II _(MilliQ)H₂O 600.0600.0 g · Kg⁻¹ Glucose 150.0 150.0 DMEM-F12m 27.0 21.0 + NaOH 10N → pH10.00 Asparagine 3.1 5.0 Yeastolate 21.2 65.0 Phytone 13.5 40.0 + HCl 5N→ pH 6.75 _(MilliQ)H₂O To 1000 g Components were weighted and added inorder to achieve 1 Kg final mass.Once prepared, both feeds I and II maintained a turbidity of 12 NTUs orless for more than 4 hrs.

Example 3.3 Cell Growth and Productivity Enhancement Due to CombinationFeed Addition

To evaluate the growth and titer promoting characteristics of the abovecombination feeds, ABT-874 cells (which express an anti IL-12fully-human IgG1 antibody) were used. This CHO cell line is normallycultivated in cell culture medium SR-383 (2× with 500 nM mtx).

For these experiments, cells were passaged into DMEM/F12 for at least 5generations until adaptation was observed by constant growth rate.Spinner cultures were stirred on a Thermolyne stir plate at 70 rpm in anincubator at 35° C. and 5% CO₂ Immediately before inoculation therequired amount of cell suspension was taken from the maintenanceculture. The cells were centrifuged, the supernatant was discarded andthe pellets were resuspended in fresh, pre-warmed medium to obtain aseed density of 4×10⁵/mL.

Cell culture was expanded in spinners until sufficient volume wasgenerated for bioreactor inoculation to achieve a split ratio of 1:5 in1.5 L Applikon bioreactors. Reactor running conditions were pH 6.9, 35°C., 150 rpm and a dissolved oxygen level of 40% of saturation. Allbioreactor experiments were performed in duplicates. Cells were giventhree 1% of initial reactor volume bolus shots of combination feed everyother day during the course of a run.

The use of both combination feeds greatly enhances cell culture growth.For the case of CF I, double the peak cell density was achieved,although the culture lasted only 10 days, as compared to 13 for thecontrol. For the case of CF II, the peak cell density was almost tripledcompared to the control and the culture lasted a similar time. In termsof final titer a more dramatic effect was observed. Titers for theDMEM/F12 medium were approximately 41 mg/L, vs 188 for CF I and 434 forCFII. The effect of different feed solutions on the maximum celldensity, culture length, titer and specific productivity is summarizedthe following table:

TABLE 25 Performance of DMEM-F12m combination feeds Parameter Peak CellDensity Final IVC Culture Final [10⁶ viable [×10⁶ Length Titer qpDescription cells/mL] cell · d/mL] [d] [mg/L] [pg/cell-d] Control (n =2) 1.07 +/− 0.018  9.53 +/− 0.005 13 41 +/− 1.9 4.7 +/− 0.29 CFI (n = 2)1.92 +/− 0.031 11.27 +/− 0.004 10 188 +/− 0.1  16.4 +/− 12.03 CFII (n =2) 2.72 +/− 0.009 20.74 +/− 0.177 13 434 +/− 16.5 25.1 +/− 1.17 

Example 3.4 High-Titer Cell Culture Processes Via Addition ofCombination Feed

Higher titers allow for fewer manufacturing runs to be needed to satisfya given total yield. The following example describes a fed-batchlarge-scale process for ABT-874 which yielded an average titer of around4 g/L during a fed-batch process. Additionally, further improvement ofthe media and combination feed allowed titer levels of over 6 g/L to beachieved.

Materials and Methods.

-   -   As a model system, the ABT-874 antibody product line was used.        Feed Solution Preparation

The feed solution was made according to the procedures previouslydescribed. Two asparagine concentrations were used, i.e., 5.0 or 7.5g·Kg⁻¹. The method of preparation is shown in the following table:

TABLE 26 ABT-874 combo feed preparation 1 _(MilliQ)H₂O 600.0 g · Kg⁻¹ 2Glucose 150.0 3 PFCHO 21.0 + 4 NaOH 10N → pH 10.00 5 Asparagine 5.0 or7.5 6 Yeastolate 65.0 7 Phytone 40.0 + 8 HCl 5N → pH 6.75 Componentswere weighted and added in order to achieve 1 Kg final mass.

Materials were dissolved in the specified mass of water using a benchtop magnetic stirrer under intense vortexing. Up to step 5, after eachcomponent was added, complete dissolution was visually verified beforethe next component was incorporated. However, this was not possible withsteps 6 and 7. For these two steps, incorporation of the powder into thesolution was considered sufficient to proceed to the final HCl addition.

Process Medium Screening

In order to obtain baseline performance data, an experiment featuring a3× Fed-Batch (FB) 3000 L as a control, the prototype 4× FB process andnon-fed (extended batch: EB) 3× and 4× conditions was run. Inparticular, it had been suggested that higher medium concentrationscreated an initial lag in the culture growth profile; however, nosignificant lag was observed for the extended batch (EB) 3× or 4×processes.

As expected, feed supplementation led to higher titers for both the 3×and 4× process. Nevertheless, significant growth suppression wasobserved for the 4× process if fed (i.e. fed-batch) Amino acid analysisof the small-scale experiments showed that, even after being fed, fulldepletion of the amino acids asparagine and glutamine still existed. Forthis reason, total feeding time and amount of asparagine in thecombination feed were both increased. In conclusion, it was determinedthat the 4× FB process had potential to reach higher final titers thanthe EB processes or the 3× FB control. Therefore, it was chosen as astarting point for further development.

Differences between the 3× fed batch process (control) and the 4× fedbatch process are described in Table 27 and described in further detailbelow.

Viable Cell Density at Feed Start

It was observed that initiating the feed at very low cell densitiestended to suppress cell growth and eventually final titer. Thus, it wasexpected that an excessively delayed feed would cause loss of volumetricproductivity due to cell starvation.

In order to investigate the above hypothesis, several experiments werecarried out to determine the significance of feeding at different viablecell densities. The combined results of those experiments are plotted inthe graph described in FIG. 1. As can be seen in FIG. 1, the titer atday 15 shows a strong dependence with the viable cell density at the daythat feeding starts. A third-degree polynomial fitted to the data pointsshows that the maximum titer on day 15 can be expected at a feedingdensity of 3.5·10⁶ cells·ml⁻¹.

Reproducibility

Process conditions for the 6 process are described below in Table 27 andwere defined as inoculation as a 1:4 split from a short fill run inSR-383, pH 7.0, DO=30%, 37° C. up to a cell density of 5.0·10⁶ viablecells/ml. Reactor running conditions were pH 6.9, T=35° C., DO=40%.Feeding was initiated when cells reached a viable density of 3.5-10⁶cells/ml, lasting 10 days, via bolus additions of combination feedconsisting of 1% of initial reactor weight each day.

Process conditions for the 4 g·L⁻¹ process are described below in Table27.

TABLE 27 Highlights of 4 and 6 g · L-1 ABT-874 cell culture processesProcess Parameter 3X FB 4X FB Medium SR-286 SR-382 (3XPFCHO) (4XPFCHO)Split ratio 1:5 1:4 Seed density 0.5-1.0 1.0-1.25 [10⁶ viable cells ·ml⁻¹] Feed Start Criterion Day 3 3.5 · 10⁶ cells · ml⁻¹ Feeding amount 11 [%] Feeding Length 7 10  [days] Asparagine in feed   5.0   7.5 [g ·l⁻¹] Temperature shift 33° C. @ none 3.5 · 10⁶ cells · ml⁻ Final Titer 4g · L⁻1 6 g · L⁻¹ ± 0.24 (n = 9)

Example 3.5 Fed-Batch Using Combination Feed Via Glucose FeedbackControl

Feedback control allows targeting a set point for a given parameter withvery limited understanding of the system intrinsic behavior. In thismanner, a targeted set point can be maintained independently of anydisturbances or alterations that the system might have undergone. Due tothe complexity of mammalian cell culture metabolism, it is laborintensive to derive comprehensive models that could allow for predictionof a culture given trajectory. Nevertheless, it is desirable to developa sampling method, e.g., by using an automated sampling, to be able tosupply glucose in order to maintain a target glucose level. This allowsdecoupling of the effect of a given glucose concentration (or othermetabolites) and also provides a means to study the effect thatdifferent ratios of glucose in the combination feed have on differentcultures. The decoupling of the effect refers to the effect ofmaintaining a given glucose level, versus the effect of using adifferent amount of glucose in a combination feed.

Materials and Methods.

As model system, the product lines for two different anti-IL-12antibodies, ABT-874 and 1D4.7 were used.

Automated sampling, i.e., YSI 2700 Bioprocess Analyzer, was chosen asthe means for monitoring glucose levels in the cell culture medium. Theonline automated sampling device was established by attaching a YSI 2730Monitor and Control Accessory to a YSI 2700 Bioprocess Analyzer (see YSILife Sciences; Yellow Springs, Ohio). This sampling device consisted ofa pump that held two tubes. The first tube had two branches, one thatcollected the sample from the bioreactor, and a second one that pumpedantiseptic to maintain sterility. Once the sample was taken, it waspumped into an external chamber, from which the sipper took the samplefor the actual analysis. The second tube through the pump was used tocollect the discharge into a waste container. Several parameters of theonline sampling accessory were controlled, such as sampling interval andTPU (Time per Unit Error, which corresponds to the time the fee pumpruns based on the measured offset from a set point).

The pump was connected to the YSI using a 15-pin connector. The pincorresponding to the glucose probe (White-7, Black-11) in the YSI wasconnected to the TTL on/off pin (8) in the pump, and one of the groundpins from the YSI (1-5) was connected to the chassis of the 15-pinconnector for the pump. The connection was tested by turning the pump onand off from the YSI Setup Menu. The pump tubing used was MasterflexCFLEX 082.

TABLE 28 Feedback initial experiment setup Cells ABT-874 Media 3XPFCHO(SR-286) Glucose Feed 400 g/L YSI TPU 16 Reactor Volume Tested 1.5 L YSIPurge Time 60 sec Pump Speed 50 (half, ~16 RPM) YSI Sampling Interval 4hrs/2 hrs (explained below) YSI Output Signal X2 (for YSI1-A25)Antiseptic 0.1N NaOH

YSI1-A25 reactor was controlled at 4.9 g/L of glucose. The glucosecontrol at 4.9 g/L started at about day 2 using a sampling interval of 4hours until day 8. At day 8, the sampling interval was reduced to 2hours, and the set point in the YSI automated device was reset to erasethe PID memory. The fluctuations due to overshoot from the set-pointwere significantly reduced after day 8. Hence, it was established that asampling interval of 2 hours was optimal for these conditions. Theaverage overshoot from day 2 to 8 was 0.43 g/L and the averageundershoot was 0.31 g/L, with a fluctuation error of about 8% from theset point. On the other hand, the average overshoot from day 8 to 13 was0.08 g/L and the average undershoot was 0.09 g/L, with a fluctuationerror of about 2%.

Control of Glucose Concentration Using Combination Feed

The schedule for feeding combination feed solutions to a culture in abioreactor was defined via an empirical approach. Different feedingamounts were tested, as were different feeding times until a viablefed-batch scheme was found. Ideally, the feeding schedule of acombination feed should meet the specific requirements of a givenculture.

In view of the above considerations, it was advantageous to provide thecombination feed based on the cell culture needs, for example, by usingglucose as an indicator of nutritional requirements. In this manner, afeedback control system may be used to 1) Test different feeds withvarying glucose concentrations and 2) Use the generated feeding profileto manually feed at the larger scale culture.

The following table summarizes two different reactor operation modesusing a cell line that produces the 1D4.7 mAb. The reference experiment(referred to as baseline in Table 29) illustrates typical titerperformance for an extended batch process in SR-372 medium, running atpH 6.9, T=35° C., DO=40% in 1.5 L Applikon bioreactors. YSI experimentswere run under the same conditions plus feedback control to supplycombination feeds containing 100, 150 or 200 g·L⁻¹ of glucose.

TABLE 29 Performance of feedback control using combination feedExperiment Final Titer [mg/L] Baseline (n = 2) 1312 +/− 33  YSI 100 (n= 1) 1974 YSI 150 (n = 2) 2044 +/− 164 YSI 200 (n = 2) 1837 +/− 163

As it can be seen from Table 29, the feedback system using a variety ofcombination feeds greatly improved final titer of the antibody.

Feeding profiles from experiments like the previous one were obtained byweighing the amount of combination feed supplied per day. A typicalfeeding profile is presented in the following table:

TABLE 30 Typical feed profile generated via feedback control (profilefor 1D4.7 antibody) Day Feed [%] 1 0.00 2 0.00 3 0.00 4 0.00 5 1.12 61.56 7 1.79 8 1.23 9 0.89 10 0.75 11 0.46 12 0.32

The above scheme can also be done to manually to feed a reactor evenwithout a feedback control system. In this manner, the feeding schedulecan be up scaled.

Summary of Results.

Fed-batch processes utilizing both a mixture of hydrolyzates and achemically defined basal medium were shown to increase the final titerof secreted mAb in mammalian cell culture.

Furthermore, a method capable of generating the following stablecombination feeds was demonstrated:

TABLE 31 Stable combination feeds D2E7 ABT-325, Feed Feed X-1 dilutedD2E7 ABT-874 I II Glucose 21 200 100, 150 150 150 g · Kg⁻¹ 150, 250PFCHO (p), 20 (p) 20 (p) 27 (p) 21 (d) 27 (d) 21 (d) DMEM- F12m (d), +NaOH 10N → pH 10.00 Asparagine 7.5 2.29 3.1 5, 7.5 3.1 5 Yeastolate 2215.7 21.2 65.0 21.2 65.0 Phytone 14 10.0 13.5 40.0 13.5 40.0 + HCl 5N →pH 6.75 _(MilliQ)H₂O To 1000 g

The combination feed described in Table 31 was made starting with_(MilliQ)H₂O (up to 750 g). As indicated above, ingredients were addedto the water to a final weight (overall weight of combination feed) of1000 g. Additionally, combination feed solutions were shown to increasecell culture longevity, peak viable cell density, and specificproductivity. Cell culture fed-batch processes using combination feedscapable of reaching titer levels up to 6 g·L⁻¹ of secreted monoclonalantibody were demonstrated. The above combination feeds were also shownto increase the cell density of the cell cultures.

Finally, a method employing a feedback control system and differentcombination feeds was also shown to be capable of increasing titerlevels. This approach could be used to accelerate cell culture processdevelopment by quickly generating feeding schedules.

REFERENCES

-   1. Whitford, W. G., Fed-Batch Mammalian Cell Culture in    Bioproduction. BioProcess International, 2006. 30-40.-   2. YSI Incorporated. (1998) YSI 2700 Select Biochemistry Analyzer    User's Manual.-   3. YSI Incorporated. (1998) YSI 2730 Monitor and Control Accessory    User's Manual.-   4. Watson Marlow Pumps. 101F, 101U User's Manual.

Example 4 Application of Sodium Butyrate and N-Acetylcysteine toIncrease the Productivity of an Anti IL-18-Producing CHO Cell Line

The present invention encompasses a novel approach to increase theproductivity of an antibody, e.g., an anti IL-18-producing CHO cellline. More specifically, the following example relates to a finalantibody, e.g., anti IL-18, titer increase via addition of chemicals tothe cell culture medium. Improvements in cell viability and antibodytiter are described below using an exemplary antibody, i.e., IL-18antibody.

Cell Line and Culture Media

The anti IL-18 antibody used in the following example is a fully humanIgG1 antibody (Ab) to IL-18. The CHO cell line expressing anti IL-18 iscultivated in a growth medium, described above in Table 4 of Example 1,SR-371. The production media for the cell line are as also describedabove in Example 1, SR-372 (used for culture in spinner flasks) andSR-382 (used for culture in bioreactors).

Culture Conditions for Experiments Carried Out in Spinner Flasks

All spinner flask experiments were performed in duplicates. The spinnercultures were stirred on a Thermolyne stir plate at 80 rpm in anincubator at 35° C. and 5% CO₂ Immediately before inoculation therequired amount of cell suspension was taken from the maintenanceculture. The cells were centrifuged, the supernatant was discarded andthe pellets were resuspended in fresh, pre-warmed culture medium toobtain a seed density of 4×10⁵/mL.

Example 4.1 Effect of Sodium Butyrate on Growth and Productivity of anAnti IL-18-Producing CHO Cell Line Cultivated in Growth Medium

To determine the concentration range of sodium butyrate, the firstexperiment was carried out in SR-371 containing various concentrationsof sodium butyrate. The experiment was carried out in 100 mL spinnerflasks with 70 mL working volume. Sodium butyrate was added from a 1 Mstock solution that was prepared by dissolving 1.101 g sodium butyratein 10 mL MilliQ water and sterilized by filtration through a 0.2 μmfilter. The solution was stored at −20° C.

Sodium Butyrate was added in the beginning of the culture (Day 0) inconcentrations of 0 mM, 0.125 mM, 0.5 mM and 1 mM. Cell density andviability were determined with an automatic cell counter (Cedex,Innovatis, Germany) in this example and all following examples. Table 32shows the viable cell density over culture time, and Table 33 describesthe viability over culture time. The experiment was carried out for 12days.

TABLE 32 Viable Cell Density over Culture Time Viable Cell Density[10⁵/ml] Flask 1: Flask 2: Flask 3: Flask 4: Flask 5: Flask 6: Flask 7:Flask 8: 0 mM 0 mM 0.125 mM 0.125 mM 0.5 mM 0.5 mM 1 mM 1 mM DayButyrate Butyrate Butyrate Butyrate Butyrate Butyrate Butyrate Butyrate0 3.2 3.85 4.46 3.01 3.66 3.98 3.83 4.07 3 36.38 33.18 27.12 29.96 16.9711.71 7.68 5.74 5 84.48 70.37 62.56 66.1 21.79 9.77 3.25 2.35 7 65.9864.19 59.15 68.65 17.72 5.41 1.04 0.7 10 7.83 11.17 8.63 11.85 4.53 6.930.8 0.61 12 1.36 2.28 3.68 3.81 2.08 1.89 0.22 0.41

TABLE 33 Viability over Culture Time Viability [%] Flask 1: Flask 2:Flask 3: Flask 4: Flask 5: Flask 6: Flask 7: Flask 8: 0 mM 0 mM 0.125 mM0.125 mM 0.5 mM 0.5 mM 1 mM 1 mM Day Butyrate Butyrate Butyrate ButyrateButyrate Butyrate Butyrate Butyrate 0 98.5 97.5 98.9 100 97.4 98.2 98.898.8 3 98.3 98.1 97.3 97.9 95.4 92.2 88.3 73.8 5 96.7 96.7 96 96.7 88.872.9 54 40.1 7 73.4 75.5 78.1 83.3 69 43.1 24.2 16.4 10 7.9 13.1 13.114.3 21 25.9 11.3 14.5 12 1.4 2.8 5.9 4.9 7.6 14.9 3.2 8.9

It can be clearly seen that sodium butyrate affected cell growth andviability. While there was no obvious effect on cell growth andviability at a butyrate concentration of 0.125 mM, there was a clearimpact on cell growth at 0.5 mM butyrate leading to a lower maximum celldensity. At 0.5 mM sodium butyrate affected viability after 5 days ofculture time. Sodium butyrate inhibited cell growth completely at aconcentration of 1 mM, and the viability decreased continuously from Day0 on.

Table 34 shows the anti IL-18 titer over culture time. The concentrationof anti IL-18 was determined by a Poros A HPLC assay in this example andall following examples.

TABLE 34 Anti IL-18 titer over culture time Titer [mg/L] Flask 1: Flask2: Flask 3: Flask 4: Flask 5: Flask 6: Flask 7: Flask 8: 0 mM 0 mM 0.125mM 0.125 mM 0.5 mM 0.5 mM 1 mM 1 mM Day Butyrate Butyrate ButyrateButyrate Butyrate Butyrate Butyrate Butyrate 0 5.4 2.4 1.8 1.6 1.3 1.31.1 1.3 3 68.9 58.1 67.7 65.7 52.1 41.9 36.3 26.2 5 173.2 150.4 169.4174.3 132.6 84.2 68.6 46.4 7 235.5 211.9 253.6 278.5 217.7 110.7 85 60.910 265.5 241.8 304.3 365.9 278.2 163.3 91.8 70.3 12 269.7 244.4 318.7385.8 282.7 175.9 94 75.4

The average final titer of the cultures with 0.125 mM butyrate was 352mg/L, the average final titer of the untreated control 257 mg/L. At thisconcentration butyrate treatment led to a 40% increase in final titer.

Example 4.2 Effect of Sodium Butyrate on Growth and Productivity of anAnti IL-18-Producing CHO Cell Line Cultivated in SR-372

The anti IL-18-expressing CHO cell line was adapted to growth in SR-372in order to exclude any possible effect of the culture split from SR-371to SR-372. All experiments with cells adapted to growth in SR-372 werecarried out in 250 mL spinner flasks with 180 mL working volume. Thecultures were carried out applying the conditions outlined above in thesection entitled “Culture Conditions for Experiments Carried out inSpinner Flasks.”

An experiment was designed to add butyrate when the cells were in themid and late exponential growth phase. The later addition of butyratewill likely cause less stress to the cells and result in improved cellgrowth and higher IVC compared to addition on Day 0 because the butyrateconcentration per cell is lower (IVC (Integral of Viable Cells) isdefined as the integral of the viable cell density versus culture time).Butyrate was added on Day 4 and Day 5 in concentrations of 0.5 mM and 2mM. The experiment was carried out for 12 days. Table 35 shows theviable cell density over culture time, Table 36 the viability overculture time. Only 2 mM sodium butyrate added on Day 4 resulted inreduced viability compared to the control, the other conditions did notaffect viability.

TABLE 35 Viable cell density over culture time Viable Cell Density[10⁵/mL] Flask 3: Flask 4: Flask 5: Flask 6: Flask 7: Flask 8: Flask 9:Flask 10: Flask 1: Flask 2: 0.5 mM 0.5 mM 2 mM 2 mM 0.5 mM 0.5 mM 2 mM 2mM 0 mM 0 mM Butyrate Butyrate Butyrate Butyrate Butyrate ButyrateButyrate Butyrate Day Butyrate Butyrate @ Day 4 @ Day 4 @ Day 4 @ Day 4@ Day 5 @ Day 5 @ Day 5 @ Day 5 0 2.35 3.37 3.08 2.61 2.27 2.41 3.2 2.272.49 2.2 3 14.51 15.79 12.79 13.69 14.04 9.42 11.41 11.36 10.42 10.93 428.88 20.91 18.88 26.11 25.48 16.09 18.78 23.15 16.57 22.16 5 28.5832.17 36.01 33.03 27.82 21.98 30.76 35.8 28.38 30.71 6 61.91 54.31 45.243.08 37.02 34.28 47.82 55.66 46.26 49.27 7 56.23 51.59 40.09 36.1933.34 27.21 42.97 52.08 41.45 48.75 10 43.06 51.89 29.2 23.33 14.0713.05 29.58 28.61 25.07 26.62 12 24.8 33.38 19.38 14.32 9.66 8.22 18.2516.18 16.01 16.93

TABLE 36 Viability over culture time Viability [%] Flask 3: Flask 4:Flask 5: Flask 6: Flask 7: Flask 8: Flask 9: Flask 10: Flask 1: Flask 2:0.5 mM 0.5 mM 2 mM 2 mM 0.5 mM 0.5 mM 2 mM 2 mM 0 mM 0 mM ButyrateButyrate Butyrate Butyrate Butyrate Butyrate Butyrate Butyrate DayButyrate Butyrate @ Day 4 @ Day 4 @ Day 4 @ Day 4 @ Day 5 @ Day 5 @ Day5 @ Day 5 0 98.4 94.6 92.7 99 95.2 94.9 98.8 97.7 96.5 98.9 3 96.6 97.298.8 99 97.2 98 97.8 97.8 96.8 97.8 4 97 98.5 99 98.6 98.5 98 98.9 97.898.7 98.2 5 96.6 97.7 97.5 97.3 96.6 97.9 98 97.2 98.1 98.3 6 96.5 96.496.2 96 93.6 92.4 96.3 96 95.9 95.8 7 95.6 95.7 93.8 92.9 87.8 87.5 94.294 92.6 91.8 10 62.6 69.4 61.9 53.9 40.8 39.2 63.7 56.9 57.7 57.1 1238.3 46.2 40.4 33.6 24.5 25.2 36.7 32.7 34.1 34.4

Table 37 reveals the anti IL-18 titer over culture time. 2 mM sodiumbutyrate added on Day 5 resulted in an increase of 29% compared to thecontrol (317 mg/L versus 245 mg/L, respectively). Cell growth was notsignificantly inhibited under these conditions (see Table 35).

TABLE 37 Anti IL-18 titer over culture time Titer [mg/L] Flask 3: Flask4: Flask 5: Flask 6: Flask 7: Flask 8: Flask 9: Flask 10: Flask 1: Flask2: 0.5 mM 0.5 mM 2 mM 2 mM 0.5 mM 0.5 mM 2 mM 2 mM 0 mM 0 mM ButyrateButyrate Butyrate Butyrate Butyrate Butyrate Butyrate Butyrate DayButyrate Butyrate @ Day 4 @ Day 4 @ Day 4 @ Day 4 @ Day 5 @ Day 5 @ Day5 @ Day 5 7 138.5 141.1 147.7 139.2 183.4 182 129.5 137.5 152.8 149.5 10192.2 208.7 226.9 203.3 269.6 259.5 202.5 205.4 266.1 260.6 12 232.2258.7 269.4 235.3 307.8 286.2 237.4 236.9 319.3 315

Example 4.3 Effect of Sodium Butyrate on Growth and Productivity of anAnti IL-18-Producing CHO Cell Line Cultivated in SR-382 in a 3 LBioreactor

The following example demonstrates an increase in final anti IL-18 titerby application of sodium butyrate to the anti IL-18 Process B (seeSection 1.5) in a large scale, i.e., 3 L bioreactors. This process wasdeveloped in 3 L Applikon bioreactors. The seed train was carried out inSR-371 until the short-fill stage (SR-372). The short-fill stage wassimulated in a 20 L Biowave Bag with 10 L working volume. The experimentinvestigating the effect of sodium butyrate on growth and productivityof the anti IL-18 Process B was carried out in 3 L Applikon bioreactorswith 1.5 L working volume. Each bioreactor was filled with 1125 mLSR-382 and inoculated by adding 375 mL of cell suspension from theBiowave Bag containing anti IL-18 cells in SR-372.

In example 4.2 the titer increase was achieved by adding butyrate whenthe cells were in the mid- to late log phase, which was day 5 in spinnerflasks. Historically, the mid- to late log phase of the anti IL-18production process in a 3 L bioreactor is on day 7 of culture time. Inthis example, day 7 was chosen for addition of sodium butyrate to theculture to ensure that butyrate is added in the mid- to late log phase.

A sodium butyrate stock solution at a concentration of 200 mM wasprepared on day 7 immediately before addition to the culture bydissolving 4.404 g sodium butyrate in 200 mL MilliQ water. This solutionwas sterilized by filtration through a 0.22 μm filter.

The experiment was carried out with 5 bioreactors. Each bioreactor runwas terminated when the respective viability was lower than 50%. Twobioreactors served as the control (anti IL-18 Process B). Sodiumbutyrate was added on day 7 of culture time to the other 3 bioreactorsin concentrations of 0.3 mM, 1 mM and 3 mM, respectively. Table 38 showsthe viable cell concentration over culture time, and Table 39 shows theviability over culture time.

TABLE 38 Viable cell density over culture time Viable Cell Density[10⁵/ml] Reactor 1: Reactor 2: Reactor 3: Reactor 4: Reactor 5: 1 mM No0.3 mM 3 mM No Day Butyrate Butyrate Butyrate Butyrate Butyrate 0 5.535.22 4.89 5.28 5.41 1 8.21 7.68 7.66 9.66 6.81 2 11.35 13.53 10.39 11.1210.07 3 15.62 17.37 12.58 14.3 13.07 4 20.69 22.4 18.39 22.46 16.49 529.58 31.55 31.15 34.03 23.43 6 41.27 44.42 43.32 46.81 32.3 7 59.0762.09 56.91 60.46 33.95 8 67.07 76.26 51.82 76.4 45.43 9 68.68 73.2363.11 61.52 57.6 10 65.55 70.17 64.53 55.52 62.45 11 66.55 72.98 66.2347.27 63.42 12 60.98 56.33 45.87 30.99 44.65 13 49.67 52.03 40.39 21.3439.1 14 38.46 30.51 21.93 18.38 15 33.23 38.37 41.34 35.97 16 27.9729.31 33.89 24.58 17 30.13 17.51 18 20.98

TABLE 39 Viability over culture time Viability [%] Reactor 1: Reactor 2:Reactor 3: Reactor 4: Reactor 5: 1 mM No 0.3 mM 3 mM No Day ButyrateButyrate Butyrate Butyrate Butyrate 0 95.3 96 92.9 94.3 96.4 1 96.4 95.496.4 96.3 95.9 2 96.3 95.9 95.9 96.1 96.7 3 96.6 95.6 97.7 96.6 95.3 497.3 97.2 96.4 95.8 95.2 5 97.1 96.4 96.2 96.4 95.3 6 96.4 96.5 96.296.8 95.9 7 96.3 95.2 95.8 94.3 95.1 8 96.2 95.2 94.2 95.1 94.8 9 95.695.2 95.4 93.6 94.2 10 94.3 94.5 94.6 87.4 93.4 11 92.2 93.9 93.8 78.993 12 88.1 91.7 92.4 51.9 92 13 80.6 86.9 90.2 33.7 89.2 14 66.7 76.288.9 84.1 15 57 60.7 85.2 73.4 16 45.9 42.7 69.5 50.7 17 58.1 34.2 18 41

The control culture grew slower in reactor 5 than in the replicate(reactor 2) due to high initial CO₂ concentrations in reactor 5. Reactor2 was terminated on day 16 of culture time (historically observed in theanti IL-18 production process), reactor 5 was terminated on day 17.Sodium butyrate at a concentration of 3 mM (reactor 4) affected cellgrowth and viability. Two days after butyrate addition cells started todie. Butyrate at 1 mM did not affect cell growth and viability (reactor1), the reactor run was terminated on day 16. Cell growth was verysimilar compared to the control in reactor 2. Butyrate at 0.3 mMprolonged the culture time for 2 days (reactor 3). Table 40 shows theanti IL-18 titer over culture time.

TABLE 40 Anti IL-18 titer over culture time Titer [mg/L] Reactor 1:Reactor 2: Reactor 3: Reactor 4: Reactor 5: 1 mM No 0.3 mM 3 mM No DayButyrate Butyrate Butyrate Butyrate Butyrate 9 1033 1074.2 905.8 966.9747.4 10 1218.4 1223.9 1096.4 1184.9 877.5 11 1410.4 1460 1261.1 1242.91053.5 12 1663.5 1538.1 1485.1 1269.5 1216.7 13 1700.2 1912.5 1750.91304.6 1303 14 1852.7 2136.9 1923.4 1429.3 15 1909.4 2223.2 2280.41672.3 16 1933.7 2324.7 2108.6 1540.5 17 2561.3 1589.4 18 2448.6

The final titer (day 16) of the culture in reactor 2 (representing antiIL-18 Process B) was 2325 g/L. The final titer in reactor 5 was 1589g/L. This lower titer is likely due to the worse cell growth caused bythe high initial CO₂ concentration in the culture medium. Butyrate at 1mM and 3 mM added on day 7 resulted in lower final titer than thecontrol (reactor 2). Butyrate at 0.3 mM added on day 7 resulted in atiter of 2561 g/L on day 17 which is an increase of 10% compared to thecontrol. This titer was the highest titer achieved in the anti IL-18process.

Example 4.4 Effect of N-Acetylcysteine (10 mM, 20 mM, 40 mM, 80 mM) onGrowth and Productivity of an Anti-IL-18-Producing CHO Cell LineCultivated in SR-372

N-Acetylcysteine can protect mammalian cells from cell death. As anantioxidant it can directly reduce reactive oxygen species. Bydeacetylation it can be converted to cysteine and increase intracellularglutathione levels. Glutathione can scavenge reactive oxygen species andserves as a substrate in the reduction of hydrogen peroxide to water.

This example demonstrates the anti IL-18 titer-increasing effect ofN-acetylcysteine. The experiments were carried out in 250 mL spinnerflasks with 180 mL working volume. The culture medium was SR-372. Beforethe experiment cells were pre-adapted to growth in SR-372 as describedin example 4.2. The spinner culture conditions were applied as describedabove in “Culture Conditions for Experiments Carried out in SpinnerFlasks.”

An N-acetylcysteine stock solution of 1 M was prepared by dissolving16.32 g N-acetylcysteine in 100 mL MilliQ water on a heated stir plate.The stock solution was sterilized by filtration through a 0.22 μmfilter. One day prior to start of the experiment N-acetylcysteine wasadded to SR-372 to obtain concentrations of 0 mM, 10 mM, 20 mM, 40 mMand 80 mM. The experiment was started by centrifugation of cells fromthe CHO anti IL-18 maintenance culture as described above in “CultureConditions for Experiments Carried out in Spinner Flasks.” Each spinnerculture was terminated when the respective viability was lower than 50%.

Cell growth was not possible in N-acetylcysteine concentrations of 20mM, 40 mM and 80 mM. Table 41 and Table 42 show the comparison of viablecell density and viability, respectively, over culture time with cellsgrown in 0 mM N-acetylcysteine and 10 mM N-acetylcysteine.

TABLE 41 Viable cell density over culture time (see top table) ViableCell Density [10⁵/mL] Flask 1: Flask 2: Flask 3: Flask 4: No N- No N- 10mM N- 10 mM N- Day acetylcysteine acetylcysteine acetylcysteineacetylcysteine 0 3.60 3.44 2.73 2.73 3 18.34 17.72 3.58 3.02 4 29.3929.71 4.47 4.24 5 33.54 31.05 6.46 6.40 6 37.38 32.92 9.84 7.27 7 34.2631.00 12.36 12.00 8 17.49 17.68 24.20 22.42 9 25.34 21.54 20.14 19.48 1017.49 17.68 24.20 22.42 11 10.75 13.84 25.02 26.00 12 27.44 27.56 1325.77 27.86 14 22.77 24.17 15 18.40 19.22 16 14.60 16.30

TABLE 42 Viability over culture time (see bottom table) Viability [%]Flask 1: Flask 2: Flask 3: Flask 4: No N- No N- 10 mM N- 10 mM N- Dayacetylcysteine acetylcysteine acetylcysteine acetylcysteine 0 97.4 96.996.2 95.3 3 98.8 98.6 57.2 52.1 4 99.0 98.8 61.9 62.2 5 98.7 98.5 69.572.1 6 95.4 92.8 76.0 73.5 7 87.8 84.0 81.5 80.9 8 73.3 69.6 85.5 82.7 964.9 60.8 82.7 86.5 10 52.5 49.0 82.5 86.9 11 38.6 38.1 81.7 87.2 1279.2 83.1 13 74.8 78.9 14 67.2 68.7 15 56.7 56.9 16 49.3 47.2

The control culture (no N-acetylcysteine) was terminated on day 11 ofculture time whereas the culture with 10 mM N-acetylcysteine could beprolonged until day 16. Initially, 10 mM N-acetylcysteine affected cellgrowth and viability and lead to a decrease in viability until day 3.Then the viability started to increase. The maximum cell density ofcultures grown in 10 mM N-acetylcysteine was lower compared to thecontrol. Table 43 demonstrates the increase in final anti IL-18 titer byN-acetylcysteine.

TABLE 43 Anti IL-18 titer over culture time Titer [mg/L] Flask 1: Flask2: Flask 3: Flask 4: No N- No N- 10 mM N- 10 mM N- Day acetylcysteineacetylcysteine acetylcysteine acetylcysteine 4 102.0 94.9 23.9 21.9 5130.5 122.4 35.3 32.5 6 168.5 154.3 60.6 55.9 7 190.4 171.8 84.2 78.0 8216.4 194.3 125.3 119.3 9 233.0 206.9 156.6 153.1 10 243.2 216.4 185.9187.7 11 258.6 227.8 224.1 230.9 12 264.1 272.6 13 300.2 311.2 14 334.0314.6 15 393.0 384.4 16 414.6 421.4

The average final titer of the control cultures is 243.2 mg/L, theaverage final titer of the cultures grown in 10 mM N-acetylcysteine was418 mg/L. This was an increase of 72% compared to the control.

Example 4.5 Effect of N-Acetylcysteine (1 mM, 2 mM, 4 mM, 8 mM) onGrowth and Productivity of an Anti-IL-18-Producing CHO Cell LineCultivated in SR-372

As described in example 4.4, N-acetylcysteine at a concentration of 10mM added on day 0 could prolong the culture time and lead to an increasein final titer. However, at this concentration cell viability initiallydecreased. Based on the results in example 4.4 an experiment wasdesigned using one tenth of the N-acetylcysteine concentrations testedin example 4.4. The conditions for this spinner flask experiment werethe same as in example 4.4. N-acetylcysteine was added in concentrationsof 0 mM (control), 1 mM, 2 mM, 4 mM and 8 mM.

Table 44 shows the viable cell density over culture time, and Table 45shows the viability over culture time.

TABLE 44 Viable cell density over culture time Flask 1: Flask 2: Flask3: Flask 4: Flask 5: 0 mM N- 0 mM N- 1 mM N- 1 mM N- 2 mM N- acetyl-acetyl- acetyl- acetyl- acetyl- Day cysteine cysteine cysteine cysteinecysteine 0 2.75 3.45 3.64 3.22 4.10 3 25.05 22.42 19.25 19.32 17.42 435.98 31.42 25.45 24.56 24.96 5 45.43 37.56 31.16 29.23 27.28 6 40.0439.41 29.67 28.35 28.45 7 35.55 33.25 27.71 27.03 26.65 8 31.56 26.2224.63 22.33 22.49 9 27.15 23.04 22.23 20.23 19.11 10 23.26 19.88 21.0617.74 18.07 11 15.48 12 13 14 Flask 6: Flask 7: Flask 8: Flask 9: Flask10: 2 mM N- 4 mM N- 4 mM N- 8 mM N- 8 mM N- acetyl- acetyl- acetyl-acetyl- acetyl- Day cysteine cysteine cysteine cysteine cysteine 0 2.963.13 2.80 2.97 3.54 3 19.03 17.39 16.39 11.24 10.17 4 28.40 24.80 23.7114.47 13.88 5 34.21 28.06 26.92 20.68 18.64 6 37.05 30.62 29.54 25.5923.13 7 32.55 30.66 26.94 28.22 26.12 8 28.99 24.75 23.84 30.00 30.77 927.75 22.25 21.71 27.45 28.63 10 23.05 20.06 19.67 27.14 27.90 11 20.7516.79 16.70 26.42 29.60 12 26.39 25.62 13 21.46 22.29 14 30.47 32.41

TABLE 45 Viability over culture time 0 mM N- 0 mM N- 1 mM N- 1 mM N- 2mM N- acetyl- acetyl- acetyl- acetyl- acetyl- cysteine cysteine cysteinecysteine cysteine 0 94.6 97.6 96.2 93.3 94.9 3 98.3 97.6 99.1 98.1 98.64 98.0 96.8 98.2 97.6 97.2 5 97.1 96.0 96.3 95.0 96.1 6 90.2 89.6 89.389.3 89.9 7 79.0 80.1 79.8 77.3 79.6 8 60.6 62.2 64.8 62.2 62.2 9 52.753.4 56.9 52.6 54.8 10 46.2 45.5 51.3 45.9 50.2 11 40.3 12 13 14 Flask6: Flask 7: Flask 8: Flask 9: Flask 10: 2 mM N- 4 mM N- 4 mM N- 8 mM N-8 mM N- acetyl- acetyl- acetyl- acetyl- acetyl- Day cysteine cysteinecysteine cysteine cysteine 0 93.8 94.9 91.7 92.1 93.6 3 97.5 97.1 98.496.4 94.3 4 98.2 97.4 97.6 96.4 94.4 5 97.1 97.1 96.9 96.5 94.4 6 93.092.4 93.1 95.8 92.0 7 86.1 84.5 85.0 93.4 89.6 8 71.9 68.4 70.6 85.684.7 9 65.2 58.5 60.3 79.7 79.5 10 54.9 52.0 55.0 76.3 75.0 11 45.1 42.545.7 68.4 69.0 12 61.3 61.2 13 51.1 52.2 14 39.4 42.6

The control cultures (0 mM N-acetylcysteine) were terminated after 10days of culture time. N-acetylcysteine could prolong culture longevity.The cultures grown in 8 mM N-acetylcysteine were terminated after 14days of culture. Cultures grown in N-acetylcysteine had a lower maximumcell density compared to the control.

Table 46 shows the effect of N-acetylcysteine on final anti IL-18 titer.

TABLE 46 Anti IL-18 titer over culture time Titer [mg/L] Flask 1: Flask2: Flask 3: Flask 4: Flask 5: 0 mM N- 0 mM N- 1 mM N- 1 mM N- 2 mM N-acetyl- acetyl- acetyl- acetyl- acetyl- Day cysteine cysteine cysteinecysteine cysteine 7 193.1 168.3 210.1 202.5 198.0 8 230.3 199.9 241.6232.2 227.5 9 244.7 216.2 256.6 245.4 242.0 10 262.7 227.2 268.9 258.3253.2 11 268.3 12 13 14 Flask 6: Flask 7: Flask 8: Flask 9: Flask 10: 2mM N- 4 mM N- 4 mM N- 8 mM N- 8 mM N- acetyl- acetyl- acetyl- acetyl-acetyl- Day cysteine cysteine cysteine cysteine cysteine 7 274.0 210.9202.0 181.9 175.1 8 323.9 251.6 240.2 237.6 232.2 9 374.7 271.7 259.3267.7 263.9 10 428.8 288.9 276.3 295.3 292.2 11 444.1 315.5 304.3 349.1367.9 12 369.5 401.4 13 429.5 432.0 14 477.6 484.4

The average final anti IL-18 titer of the control cultures was 245 mg/L(very similar to 243 mg/L in example 4.4). The final anti IL-18 titer ofthe cultures grown in 8 mM N-acetylcysteine was 481 mg/L. This is anincrease of 96% compared to the control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. The contents of allreferences, patents and published patent applications cited throughoutthis application are incorporated herein by reference.

What is claimed:
 1. A fed-batch method for producing adalimumab inmammalian cells in culture, wherein said mammalian cells express saidadalimumab, the method comprising: culturing the mammalian cells in acell culture production medium in large scale at a first temperature;and then reducing the temperature under which the mammalian cells arecultured to a second lower temperature during adalimumab production,wherein said adalimumab is produced at a titer of at least 1.3 g/L insaid cell culture and said adalimumab is further purified by a processincluding Protein A affinity chromatography.
 2. The fed-batch methodaccording to claim 1, wherein the first temperature is 35° C., 37° C.,or 38° C.
 3. The fed-batch method according to claim 1, wherein thesecond temperature is 32° C. or 33° C.
 4. The fed-batch method accordingto claim 1, wherein the first temperature is 37° C. and the secondtemperature is 33° C.
 5. The fed-batch method according to claim 1,wherein the first temperature is 37° C. and the second temperature is32° C.
 6. The fed-batch method according to claim 1, wherein the firsttemperature is 35° C. and the second temperature is 32° C.
 7. Thefed-batch method according to claim 1, wherein the cell cultureproduction medium comprises at least two different non-animal-basedhydrolysates.
 8. The fed-batch method according to claim 7, wherein thenon-animal-based hydrolysates comprise a plant-based hydrolysate and ayeast-based hydrolysate.
 9. The fed-batch method according to claim 8,wherein the plant-based hydrolysate is a soy-based hydrolysate.
 10. Thefed-batch method according to claim 1, wherein the mammalian cells areChinese Hamster Ovary (CHO) cells and the cell culture production mediumis supplemented with a 25× PFCHO solution and a 33× solution of acombination of the plant-based hydrolysate Phytone and yeast-basedhydrolysate Yeastolate.
 11. The fed-batch method according to claim 1,wherein the cell culture production medium maintains a glucoseconcentration of at least 2 g/L.
 12. The fed-batch method according toclaim 1, wherein the cell culture production medium comprises: a) amodified basal medium; b) 8 to 10 ml/kg or 110 to 130 mg/L ferriccitrate; c) 4 to 8 mL/kg or 10 to 14 mg/kg recombinant human insulin; d)5 to 9 g/kg anhydrous glucose; e) 0.1 to 1 g/kg L-glutamine; f) 1 to 3g/kg sodium bicarbonate; g) 1 to 3 g/kg HEPES; h) 2 to 3 g/kg NaCl; i)0.1 to 2 g/kg Pluronic F-68; j) 0.01 to 0.1 g/kg NaH₂PO₄—H₂O; k) 0.1 to0.1 g/kg Na₂HPO₄-7H₂O; l) 8 to 12 g/kg yeast-based hydrolysate; and m) 6to 8 g/kg plant-based hydrolysate.
 13. The fed-batch method according toclaim 1, wherein said adalimumab is produced at a titer of at least 2g/L in said cell culture.
 14. The fed-batch method according to claim13, wherein said adalimumab is produced at a titer of at least 4 g/L insaid cell culture.
 15. The fed-batch method according to claim 1,wherein the temperature is reduced after at least 3 days of culturingthe mammalian cells at the first temperature.
 16. The fed-batch methodaccording to claim 1, further comprising culturing the mammalian cellsin the cell culture production medium in large scale at a starting pH;and then reducing the pH during adalimumab production phase to a valuethat is less than the starting pH.
 17. The fed-batch method according toclaim 16, wherein the starting pH is 8 or less and the reduced pH is 6.5to 7.0.
 18. The fed-batch method according to claim 17, wherein thestarting pH is 7.1 and the reduced pH is 6.9.
 19. The fed-batch methodaccording to claim 18, wherein the pH is adjusted within the first 72hours of culturing.
 20. The fed-batch method according to claim 17,wherein the first temperature is 35° C., 37° C., or 38° C., and thesecond temperature is 32° C. or 33° C.
 21. The fed-batch methodaccording to claim 20, wherein the mammalian cells expressing adalimumabare cultured at a viable cell density of between 2.0×10⁶ cells/mL and5.0×10⁶ cells/mL.
 22. The fed-batch method according to claim 20,wherein the mammalian cells expressing adalimumab are cultured at aviable cell density of between 3.5×10⁶ cells/mL and 5.0×10⁶ cells/mL.23. The fed-batch method according to claim 17, further comprising (1)monitoring the glucose concentration in the cell culture productionmedium and (2) maintaining the glucose concentration in the cell cultureproduction medium at a concentration of at least 2 g/L, or addingglucose to the cell culture production medium when the glucoseconcentration in the cell culture production medium decreases to below 2g/L.
 24. The fed-batch method according to claim 17, further comprising(1) monitoring the glucose concentration in the cell culture productionmedium and (2) maintaining the glucose concentration in the cell cultureproduction medium at a concentration of at least 2 g/L but no greaterthan 7 g/L.
 25. The fed-batch method according to claim 17, furthercomprising (1) monitoring the glucose concentration in the cell cultureproduction medium and (2) maintaining the glucose concentration in thecell culture production medium at a concentration of at least 2 g/L butno greater than 5 g/L.
 26. The method according to claim 24, wherein theosmolarity of the cell culture production medium is 440 mOsm or less,and wherein the first temperature is 35° C., 37° C., or 38° C., and thesecond temperature is 32° C. or 33° C.
 27. The method according to claim26, wherein the cell culture production medium is maintained at between20-65% dissolved oxygen.
 28. The method according to claim 26, whereinthe cell culture production medium is maintained at about 30% dissolvedoxygen.
 29. The method according to claim 26, wherein the large-scalecell culture is greater than about 10 L.
 30. The method according toclaim 26, wherein the large-scale cell culture is 13 L.
 31. The methodaccording to claim 26, wherein the mammalian cells are Chinese HamsterOvary (CHO) cells.